Sequential protonation and deprotonation reactions govern alternating access in a Major Facilitator Superfamily proton/multidrug antiporter

Drug/proton antiporters of the Major Facilitator Superfamily are key drivers of antimicrobial resistance. However, the molecular mechanisms underlying proton-coupled drug efflux are inherently complex and remain unresolved. Here, we dissect the timing and mechanism of proton coupling in the multidrug transporter LmrP, a prototypical exporter of clinically relevant antibiotics and toxins. By directly tracking chamber opening in the native plasma membrane, we capture functionally relevant conformational transitions within a lipid environment and map them onto the catalytic carboxylates that govern transport. Our results suggest that earlier DEER distance measurements in detergent solution captured a restricted subset of conformational states, which our pH 8.0 data extend and place into a broader framework. Complementary pH 5.0 measurements reveal additional mechanistic features and, together, these datasets define the transport cycle in a membrane-embedded context. Supported by molecular dynamics simulations, the results reveal a coordinated, transport cycle-dependent mechanism in which paired catalytic carboxylates act sequentially in substrate binding, proton relay, gates control, and transporter resetting.