Bio

Academic Appointments


Honors & Awards


  • Fellow, American Academy of Microbiology (2008)
  • NIH Merit Award, NIAID/NIH (2001-2011)
  • Research Scholar, Leukemia and Lymphoma Society (2001-2006)
  • Investigator Award, Cancer Research Institute (1999-2003)
  • Pew Scholar in the Biomedical Sciences, Pew Foundation (1996-2001)

Professional Education


  • Ph.D., University of Basel, Swizterland, Biophysical Chemistry (1986)
  • B.S., Stanford University, Chemistry (1982)

Research & Scholarship

Current Research and Scholarly Interests


The Jardetzky laboratory is studying the structures and mechanisms of macromolecular complexes important in viral pathogenesis, allergic hypersensitivities and the regulation of cellular growth and differentiation, with an interest in uncovering novel conceptual approaches to intervening in disease processes. Ongoing research projects include studies of paramyxovirus and herpesvirus entry mechanisms, IgE-receptor structure and function and TGF-beta ligand signaling pathways.

Teaching

2013-14 Courses


Postdoctoral Advisees


Publications

Journal Articles


  • A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion VIROLOGY Rowe, C. L., Connolly, S. A., Chen, J., Jardetzky, T. S., Longnecker, R. 2013; 436 (1): 118-126

    Abstract

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

    View details for DOI 10.1016/j.virol.2012.10.039

    View details for Web of Science ID 000314003800014

    View details for PubMedID 23200314

  • A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions ANALYTICAL BIOCHEMISTRY Kim, B., Tarchevskaya, S. S., Eggel, A., Vogel, M., Jardetzky, T. S. 2012; 431 (2): 84-89

    Abstract

    The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, Fc?RI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged Fc?RI? proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or Fc?RI? in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-Fc?RI? dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

    View details for DOI 10.1016/j.ab.2012.09.010

    View details for Web of Science ID 000311065500002

    View details for PubMedID 22995065

  • Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor NATURE Kim, B., Eggel, A., Tarchevskaya, S. S., Vogel, M., Prinz, H., Jardetzky, T. S. 2012; 491 (7425): 613-?

    Abstract

    IgE antibodies bind the high-affinity IgE Fc receptor (Fc?RI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-Fc?RI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-Fc?RI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-Fc?RI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-Fc?RI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-Fc?RI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

    View details for DOI 10.1038/nature11546

    View details for Web of Science ID 000311339800056

    View details for PubMedID 23103871

  • Reversible Inhibition of Fusion Activity of a Paramyxovirus Fusion Protein by an Engineered Disulfide Bond in the Membrane-Proximal External Region JOURNAL OF VIROLOGY Zokarkar, A., Connolly, S. A., Jardetzky, T. S., Lamb, R. A. 2012; 86 (22): 12397-12401

    Abstract

    Cysteines were introduced into the membrane-proximal external region (MPER) of the paramyxovirus F protein. A disulfide bond formed, and the mutant protein was expressed at the cell surface but was fusion inactive. Reduction of the disulfide bond restored fusion activity. The data indicate that in addition to dissociation of the three-helix bundle stalk domain of prefusion F, the MPER region also needs to separate for F to be able to refold and cause fusion.

    View details for DOI 10.1128/JVI.02006-12

    View details for Web of Science ID 000310356400040

    View details for PubMedID 22951841

  • An Engineered Disulfide Bond Reversibly Traps the IgE-Fc(3-4) in a Closed, Nonreceptor Binding Conformation JOURNAL OF BIOLOGICAL CHEMISTRY Wurzburg, B. A., Kim, B., Tarchevskaya, S. S., Eggel, A., Vogel, M., Jardetzky, T. S. 2012; 287 (43): 36251-36257

    Abstract

    IgE antibodies interact with the high affinity IgE Fc receptor, Fc?RI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds Fc?RI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, Fc?RI?. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

    View details for DOI 10.1074/jbc.M112.407502

    View details for Web of Science ID 000310364000046

    View details for PubMedID 22948141

  • Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Welch, B. D., Liu, Y., Kors, C. A., Leser, G. P., Jardetzky, T. S., Lamb, R. A. 2012; 109 (41): 16672-16677

    Abstract

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

    View details for DOI 10.1073/pnas.1213802109

    View details for Web of Science ID 000310280300060

    View details for PubMedID 23012473

  • Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bose, S., Zokarkar, A., Welch, B. D., Leser, G. P., Jardetzky, T. S., Lamb, R. A. 2012; 109 (39): E2625-E2634

    Abstract

    The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.

    View details for DOI 10.1073/pnas.1213813109

    View details for Web of Science ID 000309604500008

    View details for PubMedID 22949640

  • Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence PLOS PATHOGENS Yuan, P., Paterson, R. G., Leser, G. P., Lamb, R. A., Jardetzky, T. S. 2012; 8 (8)

    Abstract

    Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN?) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase ?-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN? and associated reduced virulence.

    View details for DOI 10.1371/journal.ppat.1002855

    View details for Web of Science ID 000308558000032

    View details for PubMedID 22912577

  • Structure of the human metapneumovirus fusion protein with neutralizing antibody identifies a pneumovirus antigenic site NATURE STRUCTURAL & MOLECULAR BIOLOGY Wen, X., Krause, J. C., Leser, G. P., Cox, R. G., Lamb, R. A., Williams, J. V., Crowe, J. E., Jardetzky, T. S. 2012; 19 (4): 461-463

    Abstract

    Human metapneumovirus and respiratory syncytial virus cause lower respiratory tract infections. The virus fusion (F) glycoprotein promotes membrane fusion by refolding from a metastable pre-fusion to a stable post-fusion conformation. F is also a major target of the neutralizing antibody response. Here we show that a potently neutralizing anti-human metapneumovirus antibody (DS7) binds a structurally invariant domain of F, revealing a new epitope that could be targeted in vaccine development.

    View details for DOI 10.1038/nsmb.2250

    View details for Web of Science ID 000302514400017

    View details for PubMedID 22388735

  • Inhibin alpha-Subunit N Terminus Interacts with Activin Type IB Receptor to Disrupt Activin Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Zhu, J., Lin, S. J., Zou, C., Makanji, Y., Jardetzky, T. S., Woodruff, T. K. 2012; 287 (11): 8060-8070

    Abstract

    Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin ?-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin ?-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the ?-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the ?-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free ?-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (L?T2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An ?-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free ?-subunit in L?T2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the ?-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.

    View details for DOI 10.1074/jbc.M111.293381

    View details for Web of Science ID 000301349400022

    View details for PubMedID 22267736

  • The KGD Motif of Epstein-Barr Virus gH/gL Is Bifunctional, Orchestrating Infection of B Cells and Epithelial Cells MBIO Chen, J., Rowe, C. L., Jardetzky, T. S., Longnecker, R. 2012; 3 (1)

    Abstract

    Epstein-Barr virus (EBV), a member of the herpesvirus family, is the causative agent of common human infections and specific malignancies. EBV entry into target cells, including B cells and epithelial cells, requires the interaction of multiple virus-encoded glycoproteins. Glycoproteins H and L (gH/gL) cooperate with glycoprotein B (gB) to mediate fusion of the viral envelope with target cell membranes. Both the gH/gL complex and gB are required for fusion, whereas glycoprotein 42 (gp42) acts as a tropism switch and is required for B cell infection and inhibits epithelial cell infection. Our previous studies identified a prominent KGD motif located on the surface of gH/gL. In the current study, we found that this motif serves as a bifunctional domain on the surface of gH/gL that directs EBV fusion of B cells and epithelial cells. Mutation of the KGD motif to AAA decreased fusion with both epithelial and B cells and reduced the binding of gH/gL to epithelial cells and to gp42. We also demonstrate that deletion of amino acids 62 to 66 of gp42 selectively reduces binding to wild-type gH/gL, but not the KGD mutant, suggesting that the KGD motif of gH/gL interacts with the N-terminal amino acids 62 to 66 of gp42.

    View details for DOI 10.1128/mBio.00290-11

    View details for Web of Science ID 000303331400009

    View details for PubMedID 22215569

  • Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion JOURNAL OF VIROLOGY Bose, S., Welch, B. D., Kors, C. A., Yuan, P., Jardetzky, T. S., Lamb, R. A. 2011; 85 (24): 12855-12866

    Abstract

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 ?, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

    View details for DOI 10.1128/JVI.06350-11

    View details for Web of Science ID 000297642000004

    View details for PubMedID 21994464

  • Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yuan, P., Swanson, K. A., Leser, G. P., Paterson, R. G., Lamb, R. A., Jardetzky, T. S. 2011; 108 (36): 14920-14925

    Abstract

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

    View details for DOI 10.1073/pnas.1111691108

    View details for Web of Science ID 000294543400044

    View details for PubMedID 21873198

  • Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42) VIROLOGY Rowe, C. L., Matsuura, H., Jardetzky, T. S., Longnecker, R. 2011; 415 (2): 122-131

    Abstract

    The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLA class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.

    View details for DOI 10.1016/j.virol.2011.04.003

    View details for Web of Science ID 000291713500006

    View details for PubMedID 21550622

  • Fusing structure and function: a structural view of the herpesvirus entry machinery NATURE REVIEWS MICROBIOLOGY Connolly, S. A., Jackson, J. O., Jardetzky, T. S., Longnecker, R. 2011; 9 (5): 369-381

    Abstract

    Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.

    View details for DOI 10.1038/nrmicro2548

    View details for Web of Science ID 000289548800018

    View details for PubMedID 21478902

  • Mapping regions of Epstein-Barr virus (EBV) glycoprotein B (gB) important for fusion function with gH/gL VIROLOGY Plate, A. E., Reimer, J. J., Jardetzky, T. S., Longnecker, R. 2011; 413 (1): 26-38

    Abstract

    Glycoproteins gB and gH/gL are required for entry of Epstein-Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB-gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.

    View details for DOI 10.1016/j.virol.2010.12.006

    View details for Web of Science ID 000289334500004

    View details for PubMedID 21376360

  • Structure of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and TGF-beta binding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lin, S. J., Hu, Y., Zhu, J., Woodruff, T. K., Jardetzky, T. S. 2011; 108 (13): 5232-5236

    Abstract

    The zona pellucida (ZP) domain is a bipartite protein structural element comprised of ZP-N and ZP-C regions. Most notable for its ability to mediate protein polymerization, many ZP proteins polymerize and assemble into long fibrils that form specialized extracellular matrices. Other ZP proteins (namely, betaglycan and endoglin) do not polymerize but serve as important membrane coreceptors for ligands in the transforming growth factor-? (TGF-?) superfamily. Here, we present the 2.0-? resolution crystal structure of the betaglycan ZP-C region in combination with a downstream region known as the external hydrophobic patch (EHP). Similar to the ZP-N region, the ZP-C region also adopts an immunoglobulin-like fold, despite sharing no sequence homology and possessing different disulfide linkages. The EHP region, which was previously thought to be external to the ZP region, is integral to the ZP-C domain and corresponds to the ZP-C G strand. Our structure also indicates that the critical maturation cleavage of ZP proteins, a process that activates nascent ZP proteins for polymerization, occurs within the immunoglobulin domain at the FG loop. Nonpolymerizing ZP proteins such as betaglycan and endoglin do not contain this cleavage site. Finally, our structure suggests that the AB loop and the convex surface pocket are regions important for TGF-? ligand binding.

    View details for DOI 10.1073/pnas.1010689108

    View details for Web of Science ID 000288894800023

    View details for PubMedID 21402931

  • A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform ANALYTICAL BIOCHEMISTRY Park, M., Matsuura, H., Lamb, R. A., Barron, A. E., Jardetzky, T. S. 2011; 409 (2): 195-201

    Abstract

    Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no Food and Drug Administration (FDA)-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a postfusion 6-helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization (FP) assay using an engineered hRSV F protein 5-helix bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in an FP assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the "missing" sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate that this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.

    View details for DOI 10.1016/j.ab.2010.10.020

    View details for Web of Science ID 000287176600005

    View details for PubMedID 20971054

  • NMEGylation: A Novel Modification to Enhance the Bioavailability of Therapeutic Peptides BIOPOLYMERS Park, M., Jardetzky, T. S., Barron, A. E. 2011; 96 (5): 688-693

    Abstract

    We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.

    View details for DOI 10.1002/bip.21607

    View details for Web of Science ID 000295385400016

    View details for PubMedID 22180913

  • Class III Viral Membrane Fusion Proteins CELL FUSION IN HEALTH AND DISEASE II: CELL FUSION IN DISEASE Backovic, M., Jardetzky, T. S. 2011; 714: 91-101

    Abstract

    Members of class III of viral fusion proteins share common structural features and molecular architecture, although they belong to evolutionary distant viruses and carry no sequence homology. Based of the experimentally determined three-dimensional structures of their ectodomains, glycoprotein B (gB) of herpesviruses, G protein of rhabdoviruses and glycoprotein 64 (gp64) of baculoviruses have been identified as class III fusion proteins. The structures are proposed to represent post-fusion conformations, and they reveal trimeric, elongated, rod-like molecules, with each protomer being composed of five domains. Sequences which interact with target membranes and form the fusion peptides are located in two loops found at one end of the molecule. Class III fusion proteins are embedded in viral envelope with the principal function of catalyzing fusion of viral and cellular membranes, an event that is essential for infection to occur. In addition, they have been implicated in processes such as attachment to target cells and viral maturation. G protein is the only class III fusion protein for which structures of both pre- and post-fusion states have been determined, shedding light on the mechanism involved in the conformational change and membrane fusion. Whether similar structural organization of class III fusion proteins translates into a common mechanism involved in carrying out membrane fusion remains to be investigated.

    View details for DOI 10.1007/978-94-007-0782-5_3

    View details for Web of Science ID 000290775400003

    View details for PubMedID 21506008

  • Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matsuura, H., Kirschner, A. N., Longnecker, R., Jardetzky, T. S. 2010; 107 (52): 22641-22646

    Abstract

    The Epstein-Barr virus (EBV) is a ?-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.

    View details for DOI 10.1073/pnas.1011806108

    View details for Web of Science ID 000285684200055

    View details for PubMedID 21149717

  • Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays JOURNAL OF VIROLOGY Liu, F., Marquardt, G., Kirschner, A. N., Longnecker, R., Jardetzky, T. S. 2010; 84 (19): 10375-10385

    Abstract

    Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.

    View details for DOI 10.1128/JVI.00381-10

    View details for Web of Science ID 000282641800067

    View details for PubMedID 20668073

  • Structure of the Newcastle disease virus F protein in the post-fusion conformation VIROLOGY Swanson, K., Wen, X., Leser, G. P., Paterson, R. G., Lamb, R. A., Jardetzky, T. S. 2010; 402 (2): 372-379

    Abstract

    The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

    View details for DOI 10.1016/j.virol.2010.03.050

    View details for Web of Science ID 000278203800017

    View details for PubMedID 20439109

  • Characteristics of Epstein-Barr virus envelope protein gp42 VIRUS GENES Shaw, P. L., Kirschner, A. N., Jardetzky, T. S., Longnecker, R. 2010; 40 (3): 307-319

    Abstract

    Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a membrane protein essential for fusion and entry of EBV into host B-lymphocytes. Gp42 is a member of the protein-fold family C-type lectin or lectin-like domains (CLECT or CTLD) and specifically is classified as a natural-killer receptor (NKR)-like CLECT. Literature review and phylogenetic comparison show that EBV gp42 shares a common structure with other NKR-like CLECTs and possibly with many viral CTLDs, but does not appear to exhibit some common binding characteristics of many CTLDs, such as features required for calcium binding. The flexible N-terminal region adjacent to the CTLD fold is important for binding to other EBV glycoproteins and for a cleavage site that is necessary for infection of host cells. From structural studies of gp42 unbound and bound to receptor and extensive mutational analysis, a general model of how gp42 triggers membrane fusion utilizing both the flexible N-terminal region and the CTLD domain has emerged.

    View details for DOI 10.1007/s11262-010-0455-x

    View details for Web of Science ID 000276675000001

    View details for PubMedID 20162447

  • Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin alpha-Subunit, a Unique TGF beta Superfamily Antagonist PLOS ONE Zhu, J., Braun, E. L., Kohno, S., Antenos, M., Xu, E. Y., Cook, R. W., Lin, S. J., Moore, B. C., Guillette, L. J., Jardetzky, T. S., Woodruff, T. K. 2010; 5 (3)

    Abstract

    Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.

    View details for DOI 10.1371/journal.pone.0009457

    View details for Web of Science ID 000275197100003

    View details for PubMedID 20209104

  • Bimolecular Complementation of Paramyxovirus Fusion and Hemagglutinin-Neuraminidase Proteins Enhances Fusion: Implications for the Mechanism of Fusion Triggering JOURNAL OF VIROLOGY Connolly, S. A., Leser, G. P., Jardetzky, T. S., Lamb, R. A. 2009; 83 (21): 10857-10868

    Abstract

    For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the "clamp model"). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the "provocateur model"). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.

    View details for DOI 10.1128/JVI.01191-09

    View details for Web of Science ID 000270602300002

    View details for PubMedID 19710150

  • Conformational Flexibility in Immunoglobulin E-Fc(3-4) Revealed in Multiple Crystal Forms JOURNAL OF MOLECULAR BIOLOGY Wurzburg, B. A., Jardetzky, T. S. 2009; 393 (1): 176-190

    Abstract

    The structure of immunoglobulin E (IgE)-Fc(3-4) has been solved in three new crystal forms, providing 13 snapshots of the Fc conformation and revealing a diverse range of open-closed motions among subunit chains and dimers. A more detailed analysis of the open-to-closed motion of IgE-Fc(3-4) was possible with so many structures, and the new structures allow a more thorough examination of the flexibility of IgE-Fc and its implications for receptor binding. The existence of a hydrophobic pocket at the elbow region of the Fc appears to be conformation dependent and suggests a means of regulating the IgE-Fc conformation (and potentially receptor binding) with small molecules.

    View details for DOI 10.1016/j.jmb.2009.08.012

    View details for Web of Science ID 000271341400013

    View details for PubMedID 19682998

  • Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion JOURNAL OF VIROLOGY Plate, A. E., Smajlovic, J., Jardetzky, T. S., Longnecker, R. 2009; 83 (15): 7678-7689

    Abstract

    Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.

    View details for DOI 10.1128/JVI.00457-09

    View details for Web of Science ID 000267747400033

    View details for PubMedID 19457993

  • Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes JOURNAL OF VIROLOGY Sorem, J., Jardetzky, T. S., Longnecker, R. 2009; 83 (13): 6664-6672

    Abstract

    Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.

    View details for DOI 10.1128/JVI.00195-09

    View details for Web of Science ID 000267354100033

    View details for PubMedID 19369343

  • Class III viral membrane fusion proteins CURRENT OPINION IN STRUCTURAL BIOLOGY Backovic, M., Jardetzky, T. S. 2009; 19 (2): 189-196

    Abstract

    Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses, and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry.

    View details for DOI 10.1016/j.sbi.2009.02.012

    View details for Web of Science ID 000266114000011

    View details for PubMedID 19356922

  • Selectivity in the Post-Translational, Transglutaminase-Dependent Acylation of Lysine Residues BIOCHEMISTRY Murthy, S. N., Lukas, T. J., Jardetzky, T. S., Lorand, L. 2009; 48 (12): 2654-2660

    Abstract

    Transglutaminases (TGs) are known to exhibit remarkable specificities not only for the Q (or Gln) sites but also for the K (or Lys) sites of proteins with which they react. To gain further insight into K-site specificity, we examined the reactions of dansyl-epsilon-aminocaproyl-GlnGlnIleVal with three chemically and structurally well-characterized proteins (bovine pancreatic ribonuclease A, bovine pancreatic trypsin inhibitor, and chicken egg white lysozyme), as catalyzed by TG2, a biologically important post-translational enzyme. The substrates represent a total of 20 potential surface sites for acylation by the fluorescent Gln probe, yet only two of the lysine side chains reacted with TG2. While the K1 site of ribonuclease and the K15 site of the trypsin inhibitor could be readily acylated by the enzyme, none of the lysines in lysozyme were modified. The findings lead us to suggest that the selection of lysine residues by TG2 is not encoded in the primary amino acid sequence surrounding the target side chain but depends primarily on its being positioned in an accessible segment of the protein structure.

    View details for DOI 10.1021/bi802323z

    View details for Web of Science ID 000264536500009

    View details for PubMedID 19222223

  • Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Backovic, M., Longnecker, R., Jardetzky, T. S. 2009; 106 (8): 2880-2885

    Abstract

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

    View details for DOI 10.1073/pnas.0810530106

    View details for Web of Science ID 000263652900074

    View details for PubMedID 19196955

  • Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry STRUCTURE Kirschner, A. N., Sorem, J., Longnecker, R., Jardetzky, T. S. 2009; 17 (2): 223-233

    Abstract

    Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLA complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an alpha helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.

    View details for DOI 10.1016/j.str.2008.12.010

    View details for Web of Science ID 000263384800010

    View details for PubMedID 19217393

  • Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion JOURNAL OF MOLECULAR BIOLOGY Bissonnette, M. L., Donald, J. E., DeGrado, W. F., Jardetzky, T. S., Lamb, R. A. 2009; 386 (1): 14-36

    Abstract

    To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.

    View details for DOI 10.1016/j.jmb.2008.12.029

    View details for Web of Science ID 000263574300002

    View details for PubMedID 19121325

  • Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein VIROLOGY Yuan, P., Leser, G. P., Demeler, B., Lamb, R. A., Jardetzky, T. S. 2008; 378 (2): 282-291

    Abstract

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

    View details for DOI 10.1016/j.virol.2008.05.023

    View details for Web of Science ID 000258631600010

    View details for PubMedID 18597807

  • Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jing, X., Ma, C., Ohigashi, Y., Oliveira, F. A., Jardetzky, T. S., Pinto, L. H., Lamb, R. A. 2008; 105 (31): 10967-10972

    Abstract

    Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596-599] and by NMR methods [Schnell and Chou (2008) Nature 451:591-595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24-36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

    View details for DOI 10.1073/pnas.0804958105

    View details for Web of Science ID 000258308500062

    View details for PubMedID 18669647

  • Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins VIROLOGY Backovic, M., Leser, G. P., Lamb, R. A., Longnecker, R., Jardetzky, T. S. 2007; 368 (1): 102-113

    Abstract

    To gain insight into Epstein-Barr virus (EBV) glycoprotein B (gB), recombinant, secreted variants were generated. The role of putative transmembrane regions, the proteolytic processing and the oligomerization state of the gB variants were investigated. Constructs containing 2 of 3 C-terminal hydrophobic regions were secreted, indicating that these do not act as transmembrane anchors. The efficiency of cleavage of the gB furin site was found to depend on the nature of C-terminus. All of the gB constructs formed rosette structures reminiscent of the postfusion aggregates formed by other viral fusion proteins. However, substitution of putative fusion loop residues, WY(112-113) and WLIY(193-196), with less hydrophobic amino acids from HSV-1 gB, produced trimeric protein and abrogated the ability of the EBV gB ectodomains to form rosettes. These data demonstrate biochemical features of EBV gB that are characteristic of other class I and class II viral fusion proteins, but not of HSV-1 gB.

    View details for DOI 10.1016/j.virol.2007.06.031

    View details for Web of Science ID 000250679100012

    View details for PubMedID 17655906

  • Hydrophobic residues that form putative fusion loops of Epstein-Barr virus glycoprotein B are critical for fusion activity JOURNAL OF VIROLOGY Backovic, M., Jardetzky, T. S., Longnecker, R. 2007; 81 (17): 9596-9600

    Abstract

    To test the importance of the hydrophobic residues within the putative Epstein-Barr virus (EBV) glycoprotein B (gB) fusion loops in membrane fusion, WY(112-113) and WLIW(193-196) were mutated into alanine, glutamic acid, or the analogous residues from herpes simplex virus type 1 (HSV-1) gB (HR and RVEA). All gB variants exhibited cell surface expression, demonstrating that the substitutions did not perturb gB trafficking. None of six gB variants was, however, capable of mediating fusion with either epithelial or B cells. These data demonstrate that the bulky and hydrophobic EBV loop residues, which differ from the more hydrophilic HSV-1 residues and appear more compatible with membrane insertion, are essential for EBV gB-dependent fusion.

    View details for DOI 10.1128/JVI.00758-07

    View details for Web of Science ID 000248923700077

    View details for PubMedID 17553877

  • Binding-site interactions between Epstein-Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-Cell membrane fusion JOURNAL OF VIROLOGY Kirschner, A. N., Lowrey, A. S., Longnecker, R., Jardetzky, T. S. 2007; 81 (17): 9216-9229

    Abstract

    Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.

    View details for DOI 10.1128/JVI.00575-07

    View details for Web of Science ID 000248923700034

    View details for PubMedID 17581996

  • Structural basis of viral invasion: lessons from paramyxovirus F CURRENT OPINION IN STRUCTURAL BIOLOGY Lamb, R. A., Jardetzky, T. S. 2007; 17 (4): 427-436

    Abstract

    The structures of glycoproteins that mediate enveloped virus entry into cells have revealed dramatic structural changes that accompany membrane fusion and provided mechanistic insights into this process. The group of class I viral fusion proteins includes the influenza hemagglutinin, paramyxovirus F, HIV env, and other mechanistically related fusogens, but these proteins are unrelated in sequence and exhibit clearly distinct structural features. Recently determined crystal structures of the paramyxovirus F protein in two conformations, representing pre-fusion and post-fusion states, reveal a novel protein architecture that undergoes large-scale, irreversible refolding during membrane fusion, extending our understanding of this diverse group of membrane fusion machines.

    View details for DOI 10.1016/j.sbi.2007.08.016

    View details for Web of Science ID 000250423800007

    View details for PubMedID 17870467

  • Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions JOURNAL OF BIOLOGICAL CHEMISTRY Lerch, T. F., Shimasaki, S., Woodruff, T. K., Jardetzky, T. S. 2007; 282 (21): 15930-15939

    Abstract

    Follistatin (FS) regulates transforming growth factor-beta superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms. We have determined the structure of the FS315-activin A complex and quantitatively compared heparin binding by the two isoforms. The FS315 complex structure shows that both isoforms inhibit activin similarly, but FS315 exhibits movements within follistatin domain 3 (FSD3) apparently linked to binding of the C-terminal extension. Surprisingly, the binding affinities of FS288 and FS315 for heparin are similar at lower ionic strengths with FS315 binding decreasing more sharply as a function of salt concentration. When bound to activin, FS315 binds heparin similarly to the FS288 isoform, consistent with the structure of the complex, in which the acidic residues of the C-terminal extension cannot interact with the heparin-binding site. Activin-induced binding of heparin is unique to the FS315 isoform and may stimulate clearance of FS315 complexes.

    View details for DOI 10.1074/jbc.M700737200

    View details for Web of Science ID 000246589600068

    View details for PubMedID 17409095

  • The structures that underlie normal reproductive function MOLECULAR AND CELLULAR ENDOCRINOLOGY Lerch, T. F., Xu, M., Jardetzky, T. S., Mayo, K. E., Radhakrishnan, I., Kazer, R., Shea, L. D., Woodruff, T. K. 2007; 267 (1-2): 1-5

    Abstract

    The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This 'cutting edge' editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

    View details for DOI 10.1016/j.mce.2006.10.018

    View details for Web of Science ID 000245429200001

    View details for PubMedID 17140726

  • Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Connolly, S. A., Leser, G. P., Yin, H., Jardetzky, T. S., Lamb, R. A. 2006; 103 (47): 17903-17908

    Abstract

    For paramyxoviruses, two viral glycoproteins are key to the entry process: an attachment protein (HN, H, or G) and the fusion protein (F). The F protein folds to a metastable state that can be triggered to undergo large conformational rearrangements to a fusogenic intermediate and a more stable postfusion state. The triggering mechanism that controls paramyxovirus fusion has not been elucidated. To correlate the molecular structure of a soluble form of the prefusion F (PIV5 F-GCNt) with the biological function of F, soluble F protein was triggered to refold. In the absence of HN, heat was found to function as a surrogate F trigger, and F associated with liposomes and aggregated on sucrose density gradients. Electron microscopy data showed that triggered F formed rosettes. Taken together these data suggest that release and membrane insertion of the hydrophobic fusion peptide require both cleavage of F and heat. Heating of cleaved F causes conversion to a postfusion form as judged by its "golf tee" morphology in the electron microscope. Heating of uncleaved F also causes conversion of F to a morphologically similar form. The reactivity of the F protein with conformation-specific mAbs and peptide binding suggest that soluble F-GCNt and membrane-bound F proteins refold through a comparable pathway.

    View details for DOI 10.1073/pnas.0608678103

    View details for Web of Science ID 000242464900059

    View details for PubMedID 17093041

  • Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1 : 1 : 1 stable complex that acts like soluble gp42 in B-Cell fusion but not in epithelial cell fusion JOURNAL OF VIROLOGY Kirschner, A. N., Omerovic, J., Popov, B., Longnecker, R., Jardetzky, T. S. 2006; 80 (19): 9444-9454

    Abstract

    Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

    View details for DOI 10.1128/JVI.00572-06

    View details for Web of Science ID 000240647200010

    View details for PubMedID 16973550

  • The structural basis of TGF-beta, bone morphogenetic protein, and activin ligand binding REPRODUCTION Lin, S. J., Lerch, T. F., Cook, R. W., Jardetzky, T. S., Woodruff, T. K. 2006; 132 (2): 179-190

    Abstract

    The transforming growth factor-beta (TGF-beta) superfamily is a large group of structurally related growth factors that play prominent roles in a variety of cellular processes. The importance and prevalence of TGF-beta signaling are also reflected by the complex network of check points that exist along the signaling pathway, including a number of extracellular antagonists and membrane-level signaling modulators. Recently, a number of important TGF-beta crystal structures have emerged and given us an unprecedented clarity on several aspects of the signal transduction process. This review will highlight these latest advances and present our current understanding on the mechanisms of specificity and regulation on TGF-beta signaling outside the cell.

    View details for DOI 10.1530/rep.1.01072

    View details for Web of Science ID 000239925700002

    View details for PubMedID 16885528

  • Structural changes in the lectin domain of CD23, the low-affinity IgE receptor, upon calcium binding STRUCTURE Wurzburg, B. A., Tarchevskaya, S. S., Jardetzky, T. S. 2006; 14 (6): 1049-1058

    Abstract

    CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

    View details for DOI 10.1016/j.str.2006.03.017

    View details for Web of Science ID 000238730300013

    View details for PubMedID 16765898

  • Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures VIROLOGY Lamb, R. A., Paterson, R. G., Jardetzky, T. S. 2006; 344 (1): 30-37

    Abstract

    Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process-an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1-5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described.

    View details for DOI 10.1016/j.virol.2005.09.007

    View details for Web of Science ID 000234611000005

    View details for PubMedID 16364733

  • Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation NATURE YIN, H. S., Wen, X. L., Paterson, R. G., Lamb, R. A., Jardetzky, T. S. 2006; 439 (7072): 38-44

    Abstract

    Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.

    View details for DOI 10.1038/nature04322

    View details for Web of Science ID 000234378700028

    View details for PubMedID 16397490

  • Structural basis for a functional antagonist in the transforming growth factor beta superfamily JOURNAL OF BIOLOGICAL CHEMISTRY Cook, R. W., Thompson, T. B., Kurup, S. P., Jardetzky, T. S., Wookdruff, T. K. 2005; 280 (48): 40177-40186

    Abstract

    Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.

    View details for DOI 10.1074/jbc.M504591200

    View details for Web of Science ID 000233461300058

    View details for PubMedID 16186117

  • The structure of the Follistatin : Activin complex reveals antagonism of both type I and type II receptor binding DEVELOPMENTAL CELL Thompson, T. B., Lerch, T. F., Cook, R. W., Woodruff, T. K., Jardetzky, T. S. 2005; 9 (4): 535-543

    Abstract

    TGF-beta ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-beta ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

    View details for DOI 10.1016/j.devcel.2005.09.008

    View details for Web of Science ID 000232616800012

    View details for PubMedID 16198295

  • Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA YIN, H. S., Paterson, R. G., Wen, X. L., Lamb, R. A., Jardetzky, T. S. 2005; 102 (26): 9288-9293

    Abstract

    Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusion form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state.

    View details for DOI 10.1073/pnas.0503989102

    View details for Web of Science ID 000230191400037

    View details for PubMedID 15964978

  • Structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose STRUCTURE Yuan, P., Thompson, T. B., Wurzburg, B. A., Paterson, R. G., Lamb, R. A., Jardetzky, T. S. 2005; 13 (5): 803-815

    Abstract

    The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.

    View details for DOI 10.1016/j.str.2005.02.019

    View details for Web of Science ID 000229330900015

    View details for PubMedID 15893670

  • Conserved glycine residues in the fusion peptide of the paramyxovirus fusion protein regulate activation of the native state JOURNAL OF VIROLOGY Russell, C. J., Jardetzky, T. S., Lamb, R. A. 2004; 78 (24): 13727-13742

    Abstract

    Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.

    View details for DOI 10.1128/JVI.78.24.13727-13742.2004

    View details for Web of Science ID 000225409900033

    View details for PubMedID 15564482

  • Beta A versus beta B: is it merely a matter of expression? MOLECULAR AND CELLULAR ENDOCRINOLOGY Thompson, T. B., Cook, R. W., Chapman, S. C., Jardetzky, T. S., Woodruff, T. K. 2004; 225 (1-2): 9-17

    Abstract

    Activins are members of the transforming growth factor (TGF) beta (beta) superfamily of proteins that function in a wide array of physiological processes. Like other TGFbeta ligands, activins are biologically active as dimers. An activin molecule is comprised of two beta-subunits, of which four isoforms have been identified: betaA, betaB, betaC, and betaE. The most widely studied activins to date are activin A (betaA/betaA), activin B (betaB/betaB), and activin AB (betaA/betaB). Inhibin is a naturally occurring activin antagonist that consists of an alpha-subunit disulfide-linked to one of the activin beta-subunits, producing inhibin A (alpha/betaA), or inhibin B (alpha/betaB). The development of assays distinguishing between different forms of activins and inhibins, along with knock-in and knock-out models, have provided evidence that the betaA- and betaB-subunits have independent and separate roles physiologically. Additionally, evaluation of ligand-receptor interactions indicates significant differences in receptor affinity between activin isoforms, as well as between inhibin isoforms. In this review we explore the differences between activin/inhibin betaA- and betaB-subunits, including expression patterns, binding properties, and the specific structural aspects of each. From the growing pool of knowledge regarding activins and inhibins, the emerging data support the hypothesis that betaA- and betaB-subunits are functionally differently.

    View details for DOI 10.1016/j.mce.2004.02.007

    View details for Web of Science ID 000224623600003

    View details for PubMedID 15451562

  • Molecular biology of inhibin action SEMINARS IN REPRODUCTIVE MEDICINE Cook, R. W., Thompson, T. B., Jardetzky, T. S., Woodruff, T. K. 2004; 22 (3): 269-276

    Abstract

    Inhibins are dimeric glycoproteins that have primarily been studied for their role in antagonism of activin-mediated release of follicle-stimulating hormone (FSH) from gonadotropes of the anterior pituitary. As a member of the transforming growth factor beta (TGFbeta) superfamily of ligands and receptors, inhibin shares several processing and structural features with other ligands of the family. An inhibin molecule is composed of an alpha-subunit and a beta-subunit, and two isoforms have been widely investigated, inhibin A (alpha/betaA) and inhibin B (alpha/betaB). Each isoform undergoes processing from a large precursor protein to a mature 32- to 34-kDa form, depending upon the degree of glycosylation. In the absence of inhibin, for example, in ovariectomized animals or postmenopausal women, serum FSH levels rise precipitously. In unilaterally ovariectomized animals the brief loss of inhibin results in a sudden rise in FSH, which induces the remaining ovary to compensate with inhibin subunit expression in a large number of antral follicles. FSH levels are restored and the cycle continues. These studies demonstrate the need for ovarian inhibin to maintain normal gonadotropin levels. Recent studies have provided a mechanism of inhibin action that is consistent with its role in reproduction and may expand inhibin function to tissues outside the reproductive axis. Betaglycan is able to bind inhibin, and in the presence of betaglycan, the affinity of inhibin for activin receptors is increased 30-fold. Through interaction with the coreceptor, inhibin can disrupt activin interaction with its receptors and can also disrupt the interaction of activin receptors with other members of the TGFbeta superfamily, such as the bone morphogenetic proteins. These new studies provide evidence for inhibin activity in numerous organs throughout the body and for mediation of systems controlled by molecules other than activin.

    View details for Web of Science ID 000223569900014

    View details for PubMedID 15319829

  • Activation of a paramyxovirus fusion protein is modulated by inside-out signaling from the cytoplasmic tail PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Waning, D. L., Russell, C. J., Jardetzky, T. S., Lamb, R. A. 2004; 101 (25): 9217-9222

    Abstract

    Many viruses have evolved fusion-mediating glycoproteins that couple the energy released from irreversible protein refolding to the work of membrane fusion. The viral fusion proteins require a triggering event to undergo a cascade of tightly regulated conformational changes. Different isolates of the paramyxovirus SV5 fusion (F) protein have either a short (20-residue) or long (42-residue) cytoplasmic tail (CT), and a long CT suppresses fusion activity in a sequence-specific manner. Addition of a domain to the F protein CT, which has the propensity to form a three-helix bundle, stabilizes the F protein and increases the energy required for fusion activation. Quantitative cell-cell fusion assays and measurement of ectodomain conformation by monoclonal antibody reactivity indicate that this suppression of fusion by the long CT or addition of a three-helix bundle occurs at a step preceding initial membrane merger. The data suggest that F protein activation involves CT signaling to the ectodomain.

    View details for DOI 10.1073/pnas.0403339101

    View details for Web of Science ID 000222278600011

    View details for PubMedID 15197264

  • Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion JOURNAL OF VIROLOGY Silva, A. L., Omerovic, J., Jardetzky, T. S., Longnecker, R. 2004; 78 (11): 5946-5956

    Abstract

    Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.

    View details for Web of Science ID 000221513400043

    View details for PubMedID 15140992

  • Virology - A class act NATURE Jardetzky, T. S., Lamb, R. A. 2004; 427 (6972): 307-308

    View details for DOI 10.1038/427307a

    View details for Web of Science ID 000188266200028

    View details for PubMedID 14737155

  • A dual-functional paramyxovirus F protein regulatory switch segment: activation and membrane fusion JOURNAL OF CELL BIOLOGY Russell, C. J., Kantor, K. L., Jardetzky, T. S., Lamb, R. A. 2003; 163 (2): 363-374

    Abstract

    Many viral fusion-mediating glycoproteins couple alpha-helical bundle formation to membrane merger, but have different methods for fusion activation. To study paramyxovirus-mediated fusion, we mutated the SV5 fusion (F) protein at conserved residues L447 and I449, which are adjacent to heptad repeat (HR) B and bind to a prominent cavity in the HRA trimeric coiled coil in the fusogenic six-helix bundle (6HB) structure. These analyses on residues L447 and I449, both in intact F protein and in 6HB, suggest a metamorphic region around these residues with dual structural roles. Mutation of L447 and I449 to aliphatic residues destabilizes the 6HB structure and attenuates fusion activity. Mutation of L447 and I449 to aromatic residues also destabilizes the 6HB structure despite promoting hyperactive fusion, indicating that 6HB stability alone does not dictate fusogenicity. Thus, residues L447 and I449 adjacent to HRB in paramyxovirus F have distinct roles in fusion activation and 6HB formation, suggesting this region is involved in a conformational switch.

    View details for DOI 10.1083/jcb.200305130

    View details for Web of Science ID 000186331100017

    View details for PubMedID 14581458

  • Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ressing, M. E., van Leeuwen, D., Verreck, F. A., Gomez, R., Heemskerk, B., Toebes, M., Mullen, M. M., Jardetzky, T. S., Longnecker, R., Schilham, M. W., Ottenhoff, T. H., Neefjes, J., Schumacher, T. N., Hutt-Fletcher, L. M., Wiertz, E. J. 2003; 100 (20): 11583-11588

    Abstract

    Epstein-Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)-class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Valpha-domains as visualized by superimposing the crystal structures for gp42-HLA-DR1 and TCR-MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.

    View details for DOI 10.1073/pnas.2034960100

    View details for Web of Science ID 000185685700074

    View details for PubMedID 14504389

  • The IgA receptor complex: a two-for-one deal NATURE STRUCTURAL BIOLOGY Wurzburg, B. A., Jardetzky, T. S. 2003; 10 (8): 585-587

    View details for DOI 10.1080/nsb0803-585

    View details for Web of Science ID 000184412400004

    View details for PubMedID 12886289

  • Mutational analysis of the HLA class II interaction with Epstein-Barr virus glycoprotein 42 JOURNAL OF VIROLOGY McShane, M. P., Mullen, M. M., Haan, K. M., Jardetzky, T. S., Longnecker, R. 2003; 77 (13): 7655-7662

    Abstract

    Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.

    View details for DOI 10.1128/JVI.77.13.7655-7662.2003

    View details for Web of Science ID 000183598600052

    View details for PubMedID 12805465

  • Structures of an ActRIIB : activin A complex reveal a novel binding mode for TGF-beta ligand : receptor interactions EMBO JOURNAL Thompson, T. B., Woodruff, T. K., Jardetzky, T. S. 2003; 22 (7): 1555-1566

    Abstract

    The TGF-beta superfamily of ligands and receptors stimulate cellular events in diverse processes ranging from cell fate specification in development to immune suppression. Activins define a major subgroup of TGF-beta ligands that regulate cellular differentiation, proliferation, activation and apoptosis. Activins signal through complexes formed with type I and type II serine/threonine kinase receptors. We have solved the crystal structure of activin A bound to the extracellular domain of a type II receptor, ActRIIB, revealing the details of this interaction. ActRIIB binds to the outer edges of the activin finger regions, with the two receptors juxtaposed in close proximity, in a mode that differs from TGF-beta3 binding to type II receptors. The dimeric activin A structure differs from other known TGF-beta ligand structures, adopting a compact folded-back conformation. The crystal structure of the complex is consistent with recruitment of two type I receptors into a close packed arrangement at the cell surface and suggests that diversity in the conformational arrangements of TGF-beta ligand dimers could influence cellular signaling processes.

    View details for Web of Science ID 000182159500011

    View details for PubMedID 12660162

  • Structural insights into the interactions between human IgE and its high affinity receptor Fc epsilon RI MOLECULAR IMMUNOLOGY Wurzburg, B. A., Jardetzky, T. S. 2002; 38 (14): 1063-1072

    Abstract

    The interaction of IgE antibodies with the high affinity IgE receptor, FcepsilonRI, is a key step in the initiation of anti-parasitic immunity and allergic reactions. Recent structural studies of the receptor, the IgE-Fc and the IgE-Fc:FcepsilonRI complex have revealed how these two proteins interact to prime mast cell responses to antigen. The structures have revealed a novel arrangement for the FcepsilonRI ectodomains that is also observed in homologous members of this antibody receptor family. The crystal structure of the IgE-Fc:FcepsilonRI complex clarified how a 1:1 complex between the antibody and receptor is formed, with the receptor binding each chain of the antibody Fc dimer. The IgE-Fc structure in the absence of the receptor revealed the potential for large conformational rearrangements within the IgE that may affect receptor binding. These studies provide the basis for further investigation of the specificity of antibody:receptor binding and for the development of new treatments for allergic hypersensitivities.

    View details for Web of Science ID 000175986600007

    View details for PubMedID 11955598

  • Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II receptor HLA-DR1 MOLECULAR CELL Mullen, M. M., Haan, K. M., Longnecker, R., Jardetzky, T. S. 2002; 9 (2): 375-385

    Abstract

    Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

    View details for Web of Science ID 000173927000020

    View details for PubMedID 11864610

  • The analysis of the human high affinity IgE receptor Fc epsilon RI alpha from multiple crystal forms JOURNAL OF MOLECULAR BIOLOGY Garman, S. C., Sechi, S., Kinet, J. P., Jardetzky, T. S. 2001; 311 (5): 1049-1062

    Abstract

    We have solved the structure of the human high affinity IgE receptor, Fc epsilon RI alpha, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc epsilon RI alpha with its natural ligand and thus to prevent a primary step in the allergic response.

    View details for DOI 10.1006/jmbi.2001.4929

    View details for Web of Science ID 000170843200011

    View details for PubMedID 11531339

  • Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion EMBO JOURNAL Russell, C. J., Jardetzky, T. S., Lamb, R. A. 2001; 20 (15): 4024-4034

    Abstract

    Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.

    View details for Web of Science ID 000170406600015

    View details for PubMedID 11483506

  • Virus membrane fusion proteins: Biological machines that undergo a metamorphosis BIOSCIENCE REPORTS Dutch, R. E., Jardetzky, T. S., Lamb, R. A. 2000; 20 (6): 597-612

    Abstract

    Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4-3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structural elements in the processing and function of these viral fusion proteins is discussed.

    View details for Web of Science ID 000168898900011

    View details for PubMedID 11426696

  • Structure of the human IgE-Fc C epsilon 3-C epsilon 4 reveals conformational flexibility in the antibody effector domains IMMUNITY Wurzburg, B. A., Garman, S. C., Jardetzky, T. S. 2000; 13 (3): 375-385

    Abstract

    IgE antibodies mediate antiparasitic immune responses and the inflammatory reactions of allergy and asthma. We have solved the crystal structure of the human IgE-Fc Cepsilon3-Cepsilon4 domains to 2.3 A resolution. The structure reveals a large rearrangement of the N-terminal Cepsilon3 domains when compared to related IgG-Fc structures and to the IgE-Fc bound to its high-affinity receptor, FcepsilonRI. The IgE-Fc adopts a more compact, closed configuration that places the two Cepsilon3 domains in close proximity, decreases the size of the interdomain cavity, and obscures part of the FcepsilonRI binding site. IgE-Fc conformational flexibility may be required for interactions with two distinct IgE receptors, and the structure suggests strategies for the design of therapeutic compounds for the treatment of IgE-mediated diseases.

    View details for Web of Science ID 000089616400010

    View details for PubMedID 11021535

  • Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilon RI alpha NATURE Garman, S. C., Wurzburg, B. A., Tarchevskaya, S. S., Kinet, J. P., Jardetzky, T. S. 2000; 406 (6793): 259-266

    Abstract

    The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, Fc epsilonRI. Crosslinking of Fc epsilonRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with Fc epsilonRI is a key recognition event of this process and involves the extracellular domains of the Fc epsilonRI alpha-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc-Fc epsilonRI alpha complex to 3.5-A resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one C epsilon3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to Fc epsilonRI for the treatment of allergy and asthma.

    View details for Web of Science ID 000088251900038

    View details for PubMedID 10917520

  • Structural basis for paramyxovirus-mediated membrane fusion MOLECULAR CELL Baker, K. A., Dutch, R. E., Lamb, R. A., Jardetzky, T. S. 1999; 3 (3): 309-319

    Abstract

    Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.

    View details for Web of Science ID 000079459300005

    View details for PubMedID 10198633

  • Structural basis for HLA-DQ binding by the streptococcal superantigen SSA NATURE STRUCTURAL BIOLOGY Sundberg, E., Jardetzky, T. S. 1999; 6 (2): 123-129

    Abstract

    Streptococcal superantigen (SSA) is a 28,000 Mr toxin originally isolated from a pathogenic strain of Streptococcus pyogenes that has 60% sequence identity with staphylococcal enterotoxin B (SEB). SSA and SEB, however, do not compete for binding on the surfaces of cells expressing MHC class II molecules. This behavior had been ascribed to SSA and SEB binding to distinct sites on, or different subsets of, HLA-DR molecules. Here we demonstrate that SSA binds predominantly to HLA-DQ, rather than to HLA-DR molecules, and present the crystal structure of SSA at 1.85 A resolution. These data provide a structural basis for interpreting the interaction of SSA with HLA-DQ molecules as well as a foundation for understanding bacterial superantigen affinities for distinct MHC isotypes.

    View details for Web of Science ID 000078285100009

    View details for PubMedID 10048922

  • The crystal structure of the human high-affinity IgE receptor (Fc epsilon RI alpha) ANNUAL REVIEW OF IMMUNOLOGY Garman, S. C., Kinet, J. P., Jardetzky, T. S. 1999; 17: 973-976

    View details for Web of Science ID 000080436600029

    View details for PubMedID 10358779

  • Crystal structure of the human high-affinity IgE receptor CELL Garman, S. C., Kinet, J. P., Jardetzky, T. S. 1998; 95 (7): 951-961

    Abstract

    Allergic responses result from the activation of mast cells by the human high-affinity IgE receptor. IgE-mediated allergic reactions may develop to a variety of environmental compounds, but the initiation of a response requires the binding of IgE to its high-affinity receptor. We have solved the X-ray crystal structure of the antibody-binding domains of the human IgE receptor at 2.4 A resolution. The structure reveals a highly bent arrangement of immunoglobulin domains that form an extended convex surface of interaction with IgE. A prominent loop that confers specificity for IgE molecules extends from the receptor surface near an unusual arrangement of four exposed tryptophans. The crystal structure of the IgE receptor provides a foundation for the development of new therapeutic approaches to allergy treatment.

    View details for Web of Science ID 000077759100010

    View details for PubMedID 9875849

  • Alteration of a single hydrogen bond between class II molecules and peptide results in rapid degradation of class II molecules after invariant chain removal JOURNAL OF EXPERIMENTAL MEDICINE Ceman, S., Wu, S. H., Jardetzky, T. S., Sant, A. J. 1998; 188 (11): 2139-2149

    Abstract

    To characterize the importance of a highly conserved region of the class II beta chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated beta chain with a wild-type alpha chain in a murine L cell by gene transfection. The mutant class II molecule (81betaH-) assembles normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii) is coexpressed with 81betaH-, the class II-Ii complex is degraded in the endosomes. Expression of 81betaH- in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81betaH-. We propose that 81betaH- is protease sensitive because it is unable to productively associate with most peptides, including classII-associated invariant chain peptides. This model is supported by our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule and peptide are important for the integrity and stability of the complex, and that empty class II molecules are protease sensitive and degraded in endosomes. One function of DM may be to insure continuous groove occupancy of the class II molecule.

    View details for Web of Science ID 000077484700017

    View details for PubMedID 9841927

  • Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jardetzky, T. S., Brown, J. H., Gorga, J. C., Stern, L. J., Urban, R. G., STROMINGER, J. L., WILEY, D. C. 1996; 93 (2): 734-738

    Abstract

    The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.

    View details for Web of Science ID A1996TR32600038

    View details for PubMedID 8570625

  • The structure of MHC class II: a role for dimer of dimers. Seminars in immunology Schafer, P. H., Pierce, S. K., Jardetzky, T. S. 1995; 7 (6): 389-398

    Abstract

    The MHC class II molecules, expressed by antigen presenting cells, are heterodimers composed of an alpha and a beta chain, which function to present processed antigen to helper T cells. The human MHC class II molecules, HLA-DR1 and HLA-DR3, crystallized not as monomers, but rather dimers of alpha beta heterodimers. The 'dimer of dimers' or 'superdimer' structure led to speculation that the binding of T-cell receptors to monomeric class II molecules on the antigen presenting cell surface may affect dimerization and thus initiate signaling both in the T cell and in the antigen presenting cell. Recent biochemical analyses of the mouse MHC class II Ek molecule provide evidence that dimers of class II heterodimers form in the absence of T cells. Although such dimers were shown to augment T-cell stimulation, the dimerization of class II molecules alone is unlikely to initiate signal transduction. However, dimers may be important in stabilizing weak T-cell receptor/CD4/class II interactions, allowing further multimerization of such complexes, leading to signaling.

    View details for PubMedID 8775464

  • HUMAN CLASS-II MHC MOLECULE HLA-DR1 - X-RAY STRUCTURE DETERMINED FROM 3 CRYSTAL FORMS ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Brown, J. H., Jardetzky, T. S., Stern, L. J., Gorga, J. C., STROMINGER, J. L., WILEY, D. C. 1995; 51: 946-961

    Abstract

    The three-dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA-DR1, was determined to 3.3 A by X-ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 A using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen, Staphylococcus aureus enterotoxin B (SEB), bound to each DR1 molecule. Single-isomorphous replacement phasing followed by iterative two- and fourfold non-crystallographic real-space averaging between the two crystal forms resulted in 4.2 A resolution electron-density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 A for the two lymphocyte-derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexed in vitro with a specific antigenic peptide. Iterative sixfold non-crystallographic real-space averaging resulted in an electron- density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 alphabeta heterodimer in the three crystal forms are discussed.

    View details for Web of Science ID A1995TH71800009

    View details for PubMedID 15299764

  • 3-DIMENSIONAL STRUCTURE OF A HUMAN CLASS-II HISTOCOMPATIBILITY MOLECULE COMPLEXED WITH SUPERANTIGEN NATURE Jardetzky, T. S., Brown, J. H., Gorga, J. C., Stern, L. J., Urban, R. G., Chi, Y. I., Stauffacher, C., STROMINGER, J. L., WILEY, D. C. 1994; 368 (6473): 711-718

    Abstract

    The structure of a bacterial superantigen, Staphylococcus aureus enterotoxin B, bound to a human class II histocompatibility complex molecule (HLA-DR1) has been determined by X-ray crystallography. The superantigen binds as an intact protein outside the conventional peptide antigen-binding site of the class II major histocompatibility complex (MHC) molecule. No large conformational changes occur upon complex formation in either the DR1 or the enterotoxin B molecules. The structure of the complex helps explain how different class II molecules and superantigens associate and suggests a model for ternary complex formation with the T-cell antigen receptor (TCR), in which unconventional TCR-MHC contacts are possible.

    View details for Web of Science ID A1994NG55300049

    View details for PubMedID 8152483

  • CRYSTAL-STRUCTURE OF THE HUMAN CLASS-II MHC PROTEIN HLA-DR1 COMPLEXED WITH AN INFLUENZA-VIRUS PEPTIDE NATURE Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C., Urban, R. G., STROMINGER, J. L., WILEY, D. C. 1994; 368 (6468): 215-221

    Abstract

    An influenza virus peptide binds to HLA-DR1 in an extended conformation with a pronounced twist. Thirty-five per cent of the peptide surface is accessible to solvent and potentially available for interaction with the antigen receptor on T cells. Pockets in the peptide-binding site accommodate five of the thirteen side chains of the bound peptide, and explain the peptide specificity of HLA-DR1. Twelve hydrogen bonds between conserved HLA-DR1 residues and the main chain of the peptide provide a universal mode of peptide binding, distinct from the strategy used by class I histocompatibility proteins.

    View details for Web of Science ID A1994NA87000046

    View details for PubMedID 8145819

  • COMPARISON OF THE P2 SPECIFICITY POCKET IN 3 HUMAN HISTOCOMPATIBILITY ANTIGENS - HLA-A-ASTERISK-6801, HLA-A-ASTERISK-0201, AND HLA-B-ASTERISK-2705 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guo, H. C., Madden, D. R., SILVER, M. L., Jardetzky, T. S., Gorga, J. C., STROMINGER, J. L., WILEY, D. C. 1993; 90 (17): 8053-8057

    Abstract

    Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.

    View details for Web of Science ID A1993LV64400031

    View details for PubMedID 8367462

  • 3-DIMENSIONAL STRUCTURE OF THE HUMAN CLASS-II HISTOCOMPATIBILITY ANTIGEN HLA-DR1 NATURE Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., STROMINGER, J. L., WILEY, D. C. 1993; 364 (6432): 33-39

    Abstract

    The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.

    View details for Web of Science ID A1993LK81800048

    View details for PubMedID 8316295

  • DIFFERENT LENGTH PEPTIDES BIND TO HLA-AW68 SIMILARLY AT THEIR ENDS BUT BULGE OUT IN THE MIDDLE NATURE Guo, H. C., Jardetzky, T. S., Garrett, T. P., Lane, W. S., STROMINGER, J. L., WILEY, D. C. 1992; 360 (6402): 364-366

    Abstract

    We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.

    View details for Web of Science ID A1992JZ63000058

    View details for PubMedID 1448153

  • IDENTIFICATION OF SELF PEPTIDES BOUND TO PURIFIED HLA-B27 NATURE Jardetzky, T. S., Lane, W. S., Robinson, R. A., Madden, D. R., WILEY, D. C. 1991; 353 (6342): 326-329

    Abstract

    A pool of endogenous peptides bound to the human class I MHC molecule, HLA-B27, has been isolated. Microsequence analysis of the pool and of 11 HPLC-purified peptides provides information on the binding specificity of the HLA-B27 molecule. The peptides all seem to be nonamers, seven of which match to protein sequences in a database search. These self peptides derive from abundant cytosolic or nuclear proteins, such as histone, ribosomal proteins, and members of the 90K heat-shock protein family.

    View details for Web of Science ID A1991GG65400049

    View details for PubMedID 1922338

  • PEPTIDE BINDING TO HLA-DR1 - A PEPTIDE WITH MOST RESIDUES SUBSTITUTED TO ALANINE RETAINS MHC BINDING EMBO JOURNAL Jardetzky, T. S., Gorga, J. C., Busch, R., Rothbard, J., STROMINGER, J. L., WILEY, D. C. 1990; 9 (6): 1797-1803

    Abstract

    Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.

    View details for Web of Science ID A1990DF98000014

    View details for PubMedID 2189723

  • 3-DIMENSIONAL STRUCTURE OF THE BIFUNCTIONAL ENZYME N-(5'-PHOSPHORIBOSYL)ANTHRANILATE ISOMERASE-INDOLE-3-GLYCEROL-PHOSPHATE SYNTHASE FROM ESCHERICHIA-COLI PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Priestle, J. P., Grutter, M. G., WHITE, J. L., VINCENT, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., Jansonius, J. N. 1987; 84 (16): 5690-5694

    Abstract

    N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.

    View details for Web of Science ID A1987J650800037

    View details for PubMedID 3303031

  • PHOSPHORIBOSYLANTHRANILATE ISOMERASE-INDOLEGLYCEROL-PHOSPHATE SYNTHASE FROM ESCHERICHIA-COLI METHODS IN ENZYMOLOGY Kirschner, K., Szadkowski, H., Jardetzky, T. S., Hager, V. 1987; 142: 386-397

    View details for Web of Science ID A1987H889800048

    View details for PubMedID 3298981

  • DELTA HELIX - POSSIBLE LEFT-HANDED STABLE POLYPEPTIDE STRUCTURE IN THE N-TERMINAL SEGMENT OF THE LAC REPRESSOR FEBS LETTERS Chandrasekaran, R., Jardetzky, T. S., Jardetzky, O. 1979; 101 (1): 11-14

    View details for Web of Science ID A1979GV69900003

    View details for PubMedID 446720

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