Ion-Coupling of Conformational Equilibrium and Transition in Secondary Active Transporters

Abstract

In secondary active transporters, the electrochemical potential of ions across the membrane is used to fuel the “uphill” translocation of the substrate across membranes via the alternating access mechanism. The mechanism of this crucial coupling, however, is still ambiguous, despite significant recent experimental and computational progress along structural basis and ion-binding effects. Mhp1, Benzyl-hydantoin transporter, has become a key model for the secondary active transporters sharing a similar LeuT-fold topology. In the present study, we employed molecular dynamics simulations to study the impact of Na+-binding on dynamics and conformational stability of Mhp1 in multiple states and transition between them. We performed microsecond equilibrium MD simulations in outward-facing (OF) states, and biased simulations with constraint of the Na+-binding site in the inward-facing (IF) state. The simulations suggest that Na+ binding can stabilize the substrate-binding conformation in the OF state, but without a similar effect in its IF state, and reveal the underlying molecular mechanism in detail. Furthermore, the results of a special-protocol time-dependent biased simulation for state transition, suggest that Na+ binding can increase the free energy barrier along the OF-IF transition. All the results suggest that ion binding can reshape the free-energy landscape of the ion/protein complex, thereby shifting the conformational preference toward a specific OF structure, which is favorable for substrate-binding. The increased substrate affinity provided by Na+ binding will facilitate capturing the substrate from its low-concentration environment by the transporter. The rsults, therefore, provide a deeper and more comprehensive understanding for the ion-coupling mechanism of secondary active transporters.