Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, and mPAK-3

The family of p21-activated kinases (PAKs) have a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. A 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia coli was expressed to examine structure-function relationships. Results indicated that PBD binding changes the geometry of the guanine nucleotide binding site on Cdc42Hs. The catalytic domain of the GTPase activating protein, Cdc42-GAP, was found to inhibit PBD binding in a competitive manner, suggesting that this target molecule and the negative regulator (GAP) bind to overlapping sites of the Cdc42Hs molecule.