Gating Topology of the Proton-Coupled Oligopeptide Symporters

Abstract

SummaryProton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

“…S2B). The involvement of residues from TM5 and TM11 in the cytoplasmic gate of other MFS transporters (32,(37)(38)(39)(40) is consistent with this proposed role.…”

“…S2). A recent study that analyzed all available crystal structures in the MFS concluded that the cytoplasmic gate is formed by residues in TMs 4, 5, 10, and 11 (32). This led us to test the hypothesis that the above network of interactions connecting the cytoplasmic ends of TM5 and TM11 contributes to the cytoplasmic gate in VMAT2.…”

Symmetry and Structure in the POT Family of Proton Coupled Peptide Transporters

“…Essentially, the authors showed that through careful sequence alignment and structure threading, the sequence of each 3-TM inverted topology repeat unit could be transposed onto the symmetrically equivalent unit, which in the case of the MFS turns out to be A-C and B-D. The subsequent outward open state, which had previously been recalcitrant to crystallisation, was subsequently determined and shown to agree, at least qualitatively, with the "repeat-swapped" model [27]. The conceptual advance in this work was the realisation that a "symmetry-exchange" mechanism underlies the overall conformational change with the MFS and has been shown, more broadly, to operate within all currently determined secondary active transporter families [20].…”

“…Recently we used the repeat swapped methodology to propose a 'gating' topology for how POT transporters move between inward and outward facing states in response to proton and peptide binding [27]. To occlude the peptide binding site to either side of the membrane, POT transporters form gates, which are transient constrictions formed by the close packing of several transmembrane helices.…”

“…Through an analysis of the inverted topology repeats of the POT family a repeat swapped model of the outward facing state of PepT So was recently built ( Figure 3A) [27]. This model was experimentally verified using site directed spin labelling and double electron-electron resonance (DEER) spectroscopy, which measures the distance between two nitroxide spin labels attached to different helices within the protein [32].…”