Georgios Skiniotis, Postdoctoral Faculty Sponsor
Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is composed of the GTP-bound transducin alpha subunit (GalphaT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GalphaT. The structure reveals two GalphaT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEgamma subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GalphaT·GTP subunits and PDE6 that supports an alternating-site catalytic mechanism.
View details for DOI 10.1016/j.molcel.2020.09.013
View details for PubMedID 33007200
Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its alpha subunit (GalphaT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GalphaT helical domain (alphaHD) contacting the Gbetagamma subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gbetagamma during receptor-catalyzed nucleotide exchange.
View details for DOI 10.1016/j.molcel.2019.06.007
View details for PubMedID 31300275
G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and are targets for over 30% of all drugs on the market. Structural information of GPCRs and more importantly that of the complex between GPCRs and their signaling partner heterotrimeric G proteins is of great importance. Here we present a method for the large-scale purification of the rhodopsin-transducin complex, the GPCR-G protein signaling complex in visual phototransduction, directly from their native retinal membrane using native proteins purified from bovine retinae. Formation of the complex on native membrane is orchestrated in part by the proper engagement of lipid-modified rhodopsin and transducin (i.e., palmitoylation of the rhodopsin C-terminus, myristoylation and farnesylation of the αT and γ1, respectively). The resulting complex is of high purity and stability and has proved suitable for further biophysical and structural studies. The methods described here should be applicable to other recombinantly expressed receptors from insect cells or mamalian cells by forming stable, functional complexes directly on purified cell membranes.
View details for DOI 10.1007/978-1-4939-9532-5_23
View details for Web of Science ID 000487844400024
View details for PubMedID 31152413
Transmembrane proteins, such as G protein-coupled receptors (GPCR), require solubilization in detergents prior to purification. The recent development of novel detergents has allowed for the stabilization of GPCRs, which typically have a high degree of structural flexibility and are otherwise subject to denaturation. However, the detergent micelle environment is still very different from the native lipid membrane and the activity of GPCRs can be profoundly affected by interactions with annular lipid molecules. Moreover, GPCRs are often palmitoylated at their intracellular side, and a lipid bilayer environment would allow for proper orientation of these lipid modifications. Therefore, a reconstituted lipid bilayer environment would best mimic the physiological receptor microenvironment for biophysical studies of GPCRs and nanodiscs provide a methodology to address this aim. Nanodiscs are lipid bilayer discs stabilized by amphipathic membrane scaffolding proteins (MSP) where detergent-solubilized transmembrane proteins can be incorporated into them through a self-assembly process. Here we present a method for reconstituting the purified detergent-solubilized rhodopsin-transducin complex, the GPCR-G protein complex in visual phototransduction, into nanodiscs. The resulting complex incorporated into lipid nanodiscs can be used in biophysical studies including small-angle X-ray scattering and electron microscopy. This method is applicable to integral membrane proteins that mediate protein lipidation, including the zDHHC-family of S-acyltransferases and membrane-bound O-acyltransferases.
View details for DOI 10.1007/978-1-4939-9532-5_24
View details for Web of Science ID 000487844400025
View details for PubMedID 31152414
The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT-GTP and β1γ1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and β1γ1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β2-adrenergic receptor-GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.
View details for DOI 10.1074/jbc.M117.797100
View details for PubMedID 28655769
View details for PubMedCentralID PMC5572916
Human 5-lipoxygenase (5-LOX) is one of the key anti-inflammatory drug targets due to its key role in leukotrienes biosynthesis. We have built a model for the active conformation of human 5-LOX using comparative modeling, docking of known inhibitors, and molecular dynamics simulation. Using this model, novel 5-LOX inhibitors were identified by virtual screen. Of the 105 compounds tested in a cell-free assay, 30 have IC(50) values less than 100 μM and 11 less than 10 μM with the strongest inhibition of 620 nM. Compounds 4, 7, and 11 showed strong inhibition activity in the human whole blood (HWB) assay with IC(50) values of 8.6, 9.7, 8.1 μM, respectively. Moreover, compounds 4 and 7 were also found to inhibit microsomal prostaglandin E synthase (mPGES)-1 with micromolar IC(50) values, similar to licofelone, a dual functional inhibitor of 5-LOX/mPGES-1. The compounds reported here provide new scaffolds for anti-inflammatory drug design.
View details for DOI 10.1021/jm201497k
View details for Web of Science ID 000301767000008
View details for PubMedID 22380511
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