Gating topology of the proton-coupled oligopeptide symporters.
Bottom Line: Proton-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.
Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: email@example.com.Show MeSH
Mentions: The repeat-swapping method threads the sequence of repeat unit A onto the structure of repeat unit B, and vice versa, while simultaneously carrying out the same process for the C-terminal half of the protein (units C and D) (Figure 1; Figures S10 and S11), thereby creating a model in the opposing conformation, in this case outward open. The existing inward-occluded structure of PepTSo (Newstead et al., 2011) was found to be not suitable for constructing a repeat-swapped model due to asymmetries between the N- and C-terminal halves of the protein, which led to steric hindrance problems during model building. A new crystal structure of PepTSo (Protein Data Bank [PDB] ID 4UVM) at a resolution of 3.0 Å was obtained that is both more symmetric and at a higher resolution than the original structure (Figure 2A). Aligning this structure with the original PepTSo structure (PDB ID 2XUT) shows that although they are similar, i.e. inward facing, there are several key differences. First, a lateral helix between H6 and HA is resolved for the first time. A similar helix has been identified in the nitrate transporter NRT1.1 (Parker and Newstead, 2014; Sun et al., 2014). Second, the residues that make up the cytoplasmic thin gate adopt different positions (Figure 2B; Figure S2), such that this structure is inward open (the new PepTSo structure thus resembles the previously reported inward-open PepTSt structure). Third, some weak difference density (Fo − Fc) is present in a position equivalent to that observed in the previous structure, indicating that this crystal structure may not represent a fully inward-open, ligand-free state. This may explain why the structural differences are smaller than those observed for PepTSt (Lyons et al., 2014). Fourth, a solvent-accessible cavity is observed in the new structure that permits protons to access the ExxERF motif on H1 (Figure S2). This motif has previously been shown to play an important role in proton-coupled transport (Solcan et al., 2012). The repeat-swapping method was applied to both this structure of PepTSo and the existing inward-open structure of PepTSt, generating outward-facing models of both proteins (Figure 2C; Figure S2).
Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: firstname.lastname@example.org.