A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/γ-butyrobetaine antiporter CaiT.

CaiT is a homotrimeric antiporter that exchanges l-carnitine (CRN) with γ-butyrobetaine (GBB) across the bacterial membrane. Three structures have been resolved to date for CaiT, all in the inward-facing state: CRN-bound (with four CRNs per subunit), GBB-bound (two GBBs per subunit), and apo. One of the reported binding sites is the counterpart of the primary site observed in structurally similar transporters. However, the mechanism and pathway(s) of CRN/GBB unbinding and translocation, or even the ability of the substrates to dislodge from the reported binding sites, are yet to be determined. To shed light on these issues, we performed a total of 1.3 μs of molecular dynamics simulations and examined the dynamics of substrate-bound CaiT structures under different conditions. We find that both CRN and GBB are able to dissociate completely from their primary site into the cytoplasm. Substrate molecules initially located at the secondary sites dissociate even faster (within tens of nanoseconds) into the extra- or intracellular regions. Interestingly, the unbinding pathway from the primary site appears to be dictated by the geometry of the unwound part of the transmembrane (TM) helix 3, mostly around Thr(100) therein. Arg(262) on TM7, which apparently mimics the role of Na(+) in CaiT structural homologues, plays a key role in triggering the dissociation of the substrate away from the primary site and guiding its release to the cytoplasm provided that the unwound part of TM3 switches from a shielding to a yielding pose.

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