Rac1 is a small member of Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, while we focus on Rac1-PAK1 interaction by atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) experiments, we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian Network Model (GNM) analysis and Molecular Dynamics (MD) simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion suggesting the mechanical changes as potential markers of oncogenicity. Indeed, the dissociation of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1 as revealed by SMFS experiments. Q61L and Y72C mutations change the mechanics of Rac1-PAK1 complex by increasing elasticity of the protein and slow down the transition to the unbound state. On the other hand, Rac1’s intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on Switch I and II with Q61L, Y72C, oncogenic P29S and Q61R and negative T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around p-loop and Switch I decreases in all mutants, mostly in Q61L, while some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream/downstream partners including PAK1.
|Number of pages||11|
|Early online date||28 Jan 2021|
|Publication status||Published - 2 Mar 2021|