bioRxiv2022
VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.