A dock and coalesce mechanism driven by hydrophobic interactions governs Cdc42 binding with its effector protein ACK

Abstract

Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott–Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42–ACK binding.

The discovery and maturation of peptide biologics targeting the small G-protein Cdc42: A bioblockade for Ras-driven signaling

“…This is consistent with the previous observation that P7 can compete with ACK GBD binding to Cdc42 ( Figure 1C), which implies overlapping binding sites. Importantly this region of Cdc42 contributes significantly thermodynamically to the binding of both ACK and WASP (18,37) and is also demonstrated to bind the other known CRIB effectors PAK1 and Par6 where structural information is available ( Figure 6), suggesting that P7 will disrupt binding to these proteins as well.…”

“…This inhibits Cdc42-driven signalling pathways, which must be necessary for transformation by Ras in this model. This observation, together with our structural and biophysical analysis of the Cdc42-ACK GBD complex (17,18) prompted us to undertake a peptide discovery campaign, using CIS display technology, to identify a Cdc42 binding peptide that would similarly inhibit effector interaction and Ras-driven oncogenesis but would possess properties more compatible with future use as a lead therapeutic. CIS display is a biological display technology that links the DNA coding sequence of library members directly to the encoded peptide in a cell-free system by employing the binding properties of the R1 RepA protein to the origin of replication i.e.…”

“…Residues that are not accessible to the solvent are unlikely to be involved in direct interactions and their removal reduces the number of indirect contributions to chemical shift mapping. The chemical shift mapping localizes the P7 binding site to a face of Cdc42 that is involved in binding to a critical section of the N-termini of the GBDs of effector proteins (18,37).…”

CRIB effector disorder: exquisite function from chaos

“…The k on and k off rates for Cdc42-WASP are 0.793 µM -1 s -1 and 19.9 ms -1 respectively. The k on and k off rates for Cdc42-ACK are 0.224 µM -1 s -1 and 27.8 ms -1 respectively as measured by biolayer interferometry (18). Waals surface of the Cdc42-ACK structure (pdb entry 1CF4).…”

“…This hydrophobic patch interaction is further extended by Ile454 ACK (see below), which contacts Leu177, Ile46, Gly47 and Gly48 Cdc42, all of which are structurally stable. An intense concentration of hydrophobic potential is found on Cdc42, complementing the N-terminal hydrophobic residues of ACK and giving it plausible docking potential (18). This hydrophobic potential is also apparent in free Cdc42 in the GTP-bound form and so would be accessible to form an encounter complex ( Figure 5).…”