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

Closely related membrane transporters can diverge sharply in their modes of transport despite minimal sequence differences, underscoring how minor structural features can alter transport function. This divergence is exemplified in nitrate and nitrite transport 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 principles established for NarK. NarU follows a similar gating pathway but displays pronounced asymmetry favoring the outward-facing state and stabilizes an apo-occluded intermediate inaccessible to antiporters. This state arises from rotation of an arginine 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.