| Abstract
| - The small GTPase Cdc42, a member of the highly conserved Rho family of intracellularGTPases, communicates with downstream signaling proteins via high-affinity interactions with theconsensus Cdc42/Rac interactive binding (CRIB) polypeptide sequence. Previous biochemical and structuralstudies show that the CRIB motif itself is insufficient for high-affinity binding to Cdc42 but requires thesequence segment C-terminal to the CRIB motif for enhanced affinity. In this study, we have investigatedthe high-affinity (Kd in units of nanomolar) associations of two highly homologous extended CRIB domains(eCRIBs) from the PAK kinases, Cla4 and Cst20, with Cdc42 from Candida albicans. 1H−15N NMRheteronuclear NOE data of the eCRIB polypeptides in complex with Candida Cdc42 (CaCdc42) indicatethat both eCRIB peptides have approximately two binding loci for CaCdc42. When each of the two eCRIBpeptides is dissected into two fragments, the N-terminal fragments containing the minimal CRIB motif(mCRIB), mCla4 and mCst20, have relatively high binding affinities with dissociation constants (Kd) of4.2 and 0.43 μM, respectively. On the other hand, the C-terminal fragments (cCRIB), cCla4 and cCst20,exhibit significantly lower affinities for their binding to CaCdc42. The cCla4 peptide binds to CaCdc42with a sub-millimolar Kd of 275 μM, and the cCst20 peptide shows an even lower binding affinity (Kd =1160 μM). Cross-titration experiments with the cognate fragments show that the binding affinity of cCst20is enhanced ∼5.5-fold (Kd = 207 μM) in the presence of saturating amounts of mCst20, and vice versa.No such effect is observed for the binding of cCla4 and mCla4. These results suggest that the Cdc42−CRIB system can be represented by a “dual recognition” model for protein−protein interactions [Kleanthous,C., et al. (1998) Mol. Microbiol. 28, 227−233], following much the same mechanisms of multivalentmolecular interactions [Song, J., and Ni, F. (1998) Biochem. Cell Biol. 76, 177−188; Mammen, M., et al.(1998) Angew Chem., Int. Ed.37, 2754−2794]. The bivalent modeling of linked peptide fragments showsthat the binding of eCla4 follows a simple additivity/avidity model, while binding of eCst20 appears tohave a more complex mechanism involving cooperative effects. The differential binding mechanismsbetween closely related eCRIB polypeptides and CaCdc42 provide a new molecular basis for understandingkinase activation and for the design of antifungal agents targeting the large protein interaction interfacesengaged by the fungal GTPase.
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