Mechanistic Differences of Activation of Rac1(P29S) and Rac1(A159V)

[Image: see text] Rac1 is a small GTPase that plays key roles in actin reorganization, cell motility, and cell survival/growth as well as in various cancer types and neurodegenerative diseases. Similar to other Ras superfamily GTPases, Rac1 switches between active GTP-bound and inactive GDP-bound states. Switch I and II regions open and close during GDP/GTP exchange. P29S and A159V (paralogous to K-Ras(A146)) mutations are the two most common somatic mutations of Rac1. Rac1(P29S) is a known hotspot for melanoma, whereas Rac1(A159V) most commonly occurs in head and neck cancer. To investigate how these substitutions induce the Rac1 dynamics, we used atomistic molecular dynamics simulations on the wild-type Rac1 and two mutant systems (P29S and A159V) in the GTP bound state, and on the wild-type Rac1 and P29S mutated system in the GDP bound state. Here, we show that P29S and A159V mutations activate Rac1 with different mechanisms. In Rac1(P29S)-GTP, the substitution increases the flexibility of Switch I based on RMSF and dihedral angle calculations and leads to an open conformation. We propose that the open Switch I conformation is one of the underlying reasons for rapid GDP/GTP exchange of Rac1(P29S). On the other hand, in Rac1(A159V)-GTP, some of the contacts of the guanosine ring of GTP with Rac1 are temporarily lost, enabling the guanosine ring to move toward Switch I and subsequently close the switch. Rac1(A159V)-GTP adopts a Ras state 2 like conformation, where both switch regions are in closed conformation and Thr35 forms a hydrogen bond with the nucleotide.