Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange Journal Article
| Authors: | Buck, M.; Xu, W.; Rosen, M. K. |
| Article Title: | Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange |
| Abstract: | 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 × 106. 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. |
| Keywords: | signal transduction; methodology; binding affinity; protein conformation; protein domain; proteins; actin; protein binding; protein stability; gene product; enzyme activation; gene activation; amines; polymerization; peptide fragments; ligand; nuclear magnetic resonance spectroscopy; models, molecular; protons; thermodynamics; protein structure, tertiary; binding sites; protein folding; protein structure; protein family; actins; structure analysis; energy; enzyme binding; cytoskeleton; enzymes; proton transport; enzyme mechanism; protein cdc42; nitrogen isotopes; amides; amide; hydrogen; nuclear magnetic resonance, biomolecular; rho factor; guanosine triphosphatase; wiskott aldrich syndrome protein; wiskott aldrich syndrome; actin filament; cdc42 gtp-binding protein; wiskott-aldrich syndrome; wiskott-aldrich syndrome protein; priority journal; article; autoinhibitory contacts |
| Journal Title: | Biochemistry |
| Volume: | 40 |
| Issue: | 47 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 2001-11-27 |
| Start Page: | 14115 |
| End Page: | 14122 |
| Language: | English |
| DOI: | 10.1021/bi0157215 |
| PUBMED: | 11714264 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work