The Cdc42/Rac Interactive Binding Region Motif of the Wiskott Aldrich Syndrome Protein (WASP) Is Necessary but Not Sufficient for Tight Binding to Cdc42 and Structure Formation

Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescent N-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and 15N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is alpha-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.