Allosteric Regulation of Rac1-PAK1 Binding Affinity by Mutant Residues through Molecular Simulations and AFM

Abstract

Rac1 (Ras-related C3 botulinum toxin substrate 1) is a small member of Rho family of GTPases and PAK1 (p21-activated kinase 1) is a serine/threonine kinase. Switch I region is responsible for the interaction of Rac1 protein with the downstream effectors such as PAK1, whereas Switch II region is important for the upstream activation of Rac1 in its GTP-bound state. Rac1 and PAK1 regulate essential cellular processes such as cytoskeletal dynamics and actin depolymerization. Mutational activation of PAK1 by Rac1 has oncogenic signaling effects in cell proliferation, survival, invasion and metastasis. Here, we aim to explore the Rac1-PAK1 interaction to understand the allosteric regulation of the binding behavior. Q61L, on Switch II, is an oncogenic mutation leading to a constitutively active protein, whereas T17N at GTP/GDP binding site leads to either a nucleotide free or inactive protein. To this, the intrinsic dynamics of the wild type, Q61L and T17N mutant Rac1 proteins is analyzed by the Gaussian Network Model (GNM) and conventional Molecular Dynamics (MD) simulations. Preliminary results reveal that the Q61L and T17N mutations lead to altered dynamics, respectively, as stabilized fluctuations in Switch I region imply a downstream effect on PAK1 and increased fluctuations in Switch I and II regions affect both upstream and downstream interactions. The association of the Q61 and T17 positions with the global hinge sites supports the allosteric coupling between the mutations and Rac1-PAK1 binding. The Y72C oncogenic mutation at a hinge site is to be analyzed in this framework with respect to the binding dynamics of Rac1 and functional implications in oncogenic signaling. The effects of these three mutations on the free energy landscape of Rac1-PAK1 are then to be explored and verified via Atomic Force Microscopy (AFM).