Proton hopping and secondary gating reveal a universal transport mechanism in a minimal bacterial transporter EmrE.

The alternating access model has been widely applied to help understand many secondary transporters, but there are emerging exceptions that question the universal validity of such a theory. EmrE is a small multidrug resistant (SMR) transporter that utilizes the proton gradient across the membrane to efflux drug molecules from the cell. Recent experimental evidence has suggested that EmrE does not meet the conditions required for the alternating access model but still achieves coupled transport. This, consequently, suggests the need for a universal exchange model to explain the coupled transport. In this paper, through a combined computational and experimental approach, we provide key evidence that the transport cycle of EmrE follows the universal exchange model. First, we show that a proton can move between the two drug/proton co-binding glutamic acid residues (E14s) in proximity to one another ("proton hopping") at a rate faster than alternating access, which is a key step in the universal exchange model. Furthermore, we show that when both sites are bound with protons, two peripheral residues E25 and T56 will coordinate to occlude EmrE to prevent proton leak. The proposed model rectifies outstanding differences in the current mechanistic understanding of promiscuous SMR transporters and defines a means of maintaining a high degree of coupling with increased substrate promiscuity.