The Structural Basis for Cdc42-Induced Dimerization of IQGAPs.
2016; 24 (9): 1499-1508
Conserved sequence repeats of IQGAP1 mediate binding to Ezrin.
Journal of proteome research
2014; 13 (2): 1156-1166
In signaling, Rho-family GTPases bind effector proteins and alter their behavior. Here we present the crystal structure of Cdc42·GTP bound to the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP2. Four molecules of Cdc42 are bound to two GRD molecules, which bind each other in a parallel dimer. Two Cdc42s bind very similarly to the Ras/RasGAP interaction, while the other two bind primarily to "extra domain" sequences from both GRDs, tying the GRDs together. Calorimetry confirms two-site binding of Cdc42·GTP for the GRDs of both IQGAP2 and IQGAP1. Mutation of important extra domain residues reduces binding to single-site and abrogates Cdc42 binding to a much larger IQGAP1 fragment. Importantly, Rac1·GTP displays only single-site binding to the GRDs, indicating that only Cdc42 promotes IQGAP dimerization. The structure identifies an unexpected role for Cdc42 in protein dimerization, thus expanding the repertoire of interactions of Ras family proteins with their targets.
View details for DOI 10.1016/j.str.2016.06.016
View details for PubMedID 27524202
Loop 5-directed Compounds Inhibit Chimeric Kinesin-5 Motors IMPLICATIONS FOR CONSERVED ALLOSTERIC MECHANISMS
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (8): 6201-6210
Mammalian IQGAP proteins all feature multiple ∼50 amino acid sequence repeats near their N-termini, and little is known about the function of these "Repeats". We have expressed and purified the Repeats from human IQGAP1 to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin, and moesin. ERM proteins have an N-terminal FERM domain (4.1, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2) and a C-terminal actin-binding domain and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain, while no binding is seen for full-length "autoinhibited" ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains for signaling purposes.
View details for DOI 10.1021/pr400787p
View details for PubMedID 24294828
pH induces thermal unfolding of UTI: An implication of reversible and irreversible mechanism based on the analysis of thermal stability, thermodynamic, conformational characterization
JOURNAL OF FLUORESCENCE
2008; 18 (2): 305-317
The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.
View details for DOI 10.1074/jbc.M110.154989
View details for Web of Science ID 000287476400029
View details for PubMedID 21127071
The thermal unfolding of Urinary Trypsin Inhibitor (UTI) was studied by several methods: Circular Dichroism (CD), Fluorescence and UV-Vis spectra. Thermal melting of UTI, dissolved in the neutral and basic buffers, was proved to be irreversible and two domains of UTI unfolded simultaneously, but the melting was reversible and the intermediate was observed when pH is lower than 4.2. The result suggested that heat and changes in pH, which had a more important impact on the stabilization of the domain I and the interaction between two domains, might cause different unfolding transitions. A reasonable explanation was deduced for the mechanism of reversible and irreversible thermal unfolding based on the effect of pH on the protein structure, the analysis of thermal transitions and the result of Electron Microscopy: In neutral and basic buffers, the Reactive Central Loop (RCL) in domain II can interact with or insert into the partial expanding domain I and UTI become self-polymerization, however, no aggregation can be observed in acid buffer since low pH and heat destabilized the structure of the domain I and the native conformation can restructure. The interaction between the special structural element RCL and domain I play an important role in the formation of polymer which was different from other two reasons given by other authors--the cleavage of disulfide and the formation of irregular polymer mainly based on hydrophobic interaction.
View details for DOI 10.1007/s10895-007-0270-5
View details for Web of Science ID 000253896400009
View details for PubMedID 17992566