Abstract
Secondary active transporters harness the electrochemical gradient of one of the substrates, typically H+ or Na+, to translocate molecules or ions across the cell membrane. They use an alternating-access mechanism to couple these downhill and uphill transport effectively. However, despite years of investigation, some fundamental aspects of this process still need to be elucidated. Namely, it remains to be determined the structure and energetics of relevant intermediate protein conformations along the transport cycle and how substrate binding alters them to ensure selective and active transport.
In this work, we resolve these questions for a prokaryotic homolog of the well-known cardiac Na+/Ca2+ exchanger, using enhanced-sampling all-atom simulations and an original method to translate trajectory data into free-energy landscapes. We base these simulations on the available X-ray structure of the protein, which so far could be resolved only in the outward-facing state. After assessing the good convergence of the simulation data and their consistency with previous crystallographic data, we corroborate the results against available solvent-accessibility and hydrogen-deuterium exchange (HDX) measurements; the latter are examined also using a novel methodology.
The results reveal the structural features of the inward-facing conformation of the transporter and provide direct evidence for a new type of mechanism. During the alternating-access transition, ions remain bound in the center of the protein. At the same time, two pseudo-symmetric helices slide across the lipid bilayer, opening a pathway to the binding sites from one side of the membrane while occluding access from the other. Additional simulations demonstrate that the alternating access transition requires the binding of either three Na+ or one Ca+ ion, as only in these occupancy states the occluded intermediate conformations are energetically accessible. Overall, our results explain at the molecular level the emergence of selective and active transport in this class of transporters, in agreement with the well-established 3Na+:1Ca+ stoichiometry.
Significance Membrane transporters regulate critical cellular processes, ranging from substrate recruitment into the cell to ions homeostasis and signaling. Therefore, besides the general interest in rationalizing the function of these proteins, understanding their molecular mechanisms in detail is essential to identify possible inhibitors or stimulators that can be used as therapeutic agents for pathophysiological conditions. Here we used quantitative molecular simulation methods to elucidate the molecular steps of the transport cycle of a Na+/Ca2+ exchanger (NCX). This protein pertains to a prominent family of membrane transporters involved in cardiac diseases. The simulation results, validated with previous biophysical and biochemical experiments, provide new fundamental insight into the transport process of the NCX family, explaining at the molecular level their known selectivity and exchange stoichiometry. Hence, the emergent mechanism presented here expands our understanding of the basic principles governing the function of secondary active transporters.
Competing Interest Statement
The authors have declared no competing interest.