Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.
The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.
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Howard Hughes Medical Institute, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA.
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Rosen
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D000199:Actins / Q000378:metabolism; D000577:Amides; D001665:Binding Sites; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011522:Protons; D013816:Thermodynamics; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D000577:Amides; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D011522:Protons; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein