How minor sequence changes enable mechanistic diversity in MFS transporters? An atomic-level rationale for symport emergence in NarU

Abstract Closely related membrane transporters can diverge sharply in their modes of trans-port despite minimal sequence differences, underscoring how minor structural features can alter transport function. This divergence is exemplified in nitrate and nitrite trans-port across bacterial membranes, which supports anaerobic respiration and involves the major facilitator superfamily (MFS) transporters NarK and NarU. NarK operates as a nitrate/nitrite antiporter, whereas NarU’s mechanism remains unresolved, with evidence suggesting potential symport activity. Using extensive adaptive molecular dynamics simulations and Markov State Modeling, we mapped NarU’s conformational free-energy landscape and assessed how its behavior contrasts with mechanistic prin-ciples established for NarK. NarU follows a similar gating pathway but displays pro-nounced asymmetry favoring the outward-facing state and stabilizes an apo -occluded intermediate inaccessible to antiporters. This state arises from rotation of an argi-nine gating pair and a hinge glycine substitution that enhances gate flexibility. These sequence-dependent adaptations alter gating energetics and reprogram the scaffold to permit coupled co-transport. Our results show that a presence of a few strategic residue substitutions in the binding pocket and translocation pathway could alter the transport mechanism of transporters with high sequence and structural similarity.