Influence of a Ser111-phosphorylation on Rab1b GTPase conformational dynamics studied by advanced sampling simulations

Rab GTPases constitute the largest branch of the Ras protein superfamily that regulate intra-cellular membrane trafficking. Their signaling activity is mediated by the transition between an active GTP-bound state and an inactive GDP-bound state. In the inactive state the switch I and II segments adopt largely disordered flexible conformations, whereas in the active state these regions are in well-defined conformations. The switch I and II states are central for recognition of Rab GTPases by interacting partners. Phosphorylation of the Rab1b-GTPase at residue Ser111 (pS111) results in modulation of the signaling activity due to alterations of the protein interaction interface and also due to modulation of the conformational flexibility. We have studied the flexibility of native and pS111-Rab1b in complex with GTP or GDP using extensive Molecular Dynamics (MD) simulations and an advanced sampling method called DIhedral Angle-biasing potential Replica-Exchange Molecular dynamics (DIA-REMD). The DIA-REMD method promotes backbone and side chain dihedral transitions along a series of replica simulations in selected protein segments and through exchanges also improves sampling in an unbiased reference simulation. Application to the Rab1b system results in significantly enhanced sampling of different switch I/II conformational states in the GDP-bound Rab1b state. The pS111 modification is found to reduce the conformational flexibility even in the presence of GDP, which may influence signaling activities. The stabilizing effect can be attributed to the formation of additional surface salt bridges between Arg-residues and pS111 not present in the native structure. The DIA-REMD method could be a valuable approach for studying also other signaling proteins that contain flexible segments.