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.
Highlights
Peptide transport is the main route through which the body absorbs and retains dietary protein and plays an important role in human physiology (Steinhardt and Adibi, 1986)
Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters
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
Summary
Peptide transport is the main route through which the body absorbs and retains dietary protein and plays an important role in human physiology (Steinhardt and Adibi, 1986). The resulting diand tripeptides are actively transported across the intestinal brush border membrane by the integral membrane peptide transporter, PepT1 (Fei et al, 1994; Leibach and Ganapathy, 1996). PepT1 recognizes a diverse range of small peptides and is responsible for the absorption of many orally administered drugs, including b-lactam antibiotics and a growing number of peptiditic prodrugs (Luckner and Brandsch, 2005; Pieri et al, 2009; Brandsch, 2009). PepT1 is a member of the POT family of proton-dependent oligopeptide transporters (TC 2.A.17), which itself belongs to the much larger major facilitator superfamily (MFS) of secondary active transport proteins (Reddy et al, 2012). Biochemical studies on the bacterial POT family have revealed that these proteins operate in a similar way to their mammalian counterparts, with many of the functionally important residues conserved (Figure S8) (Daniel et al, 2006; Harder et al, 2008)
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