Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2

E Sila Ozdemir,Hyunbum Jang,Attila Gursoy,Ozlem Keskin,Zhigang Li,David B Sacks,Ruth Nussinov

doi:10.1074/jbc.ra117.001596

Abstract

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

Highlights

IQ motif– containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell– cell adhesion, polarity, and motility
Cdc42 insert-loop binding to the Ex-domain induces allosteric changes in the RasGAP site that facilitate GRD dimerization
Root mean square fluctuation (RMSF) calculations show that the residues in C-term fluctuate much more than the other residues of GRD of IQGAP2 (GRD2) upon Ex-mode Cdc42 binding

Summary

The abbreviations used are

CaM, calmodulin; IQGAP, IQ motif– containing GTPase-activating protein; WASP, Wiskott–Aldrich syndrome protein; MD, molecular dynamics; C-term, C-terminal region of GRD2; RMSF, root mean square fluctuation; RMSD, root mean square deviation; WISP, weighted implementation of the suboptimal paths; H-bond, hydrogen bond; MM, molecular mechanics energies; GBSA, generalized Born surface area continuum solvation; CRIB, Cdc42/Rac binding; PVDF, polyvinylidene difluoride; PDB, Protein Data Bank. Some of the binding partners compete or interfere with each other for binding to IQGAPs. For example, CaM binding causes allosteric Cdc release from GRD and decreases actin binding to the calponin homology domain [34]. It was speculated that only one Rac molecule can bind to GRD, and there has been no evidence that it facilitates IQGAP dimerization [16]; the exact mechanism of Rac1GRD2 interaction and the differing stoichiometry have not been identified. We observed that the insert loop and switch I of Cdc are mainly responsible for Cdc binding to the Ex-domain This binding releases the Ex-domain at the C-terminal region of GRD2 (hereafter referred to as C-term) to facilitate dimerization. We unravel the underlying mechanism of the different stoichiometry that is involved in the binding of Cdc and Rac to GRD and of IQGAP2 dimerization. We suggest how the mechanism can help to elucidate the key question of the roles of Cdc and Rac in actin polymerization in metastasis

Results

Discussion

Experimental procedures