Membrane Chemistry Tunes the Structure of a Peptide Transporter

Abstract

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities,high-resolution structures are usually obtained in artificial detergents.T ou nderstand whether and how lipids guide membrane protein function, we use single-molecule FRET to probe the dynamics of DtpA, amember of the proton-coupled oligopeptide transporter (POT) family,i nv arious lipid environments.W es how that detergents trap DtpA in ad ynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity,a llowing an opening to the extracellular side and asampling of all relevant states.Bilayer compositions tune the abundance of these states. Anovel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups.Hence,chemical diversity of membranes translates into structural diversity,w ith the current POTs tructures only sampling ap ortion of the full structural space.

Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

“…Nevertheless, all structures of bacterial homologues (47 Protein Databank (PDB) entries representing ten different bacterial POTs) have been captured in the inward-open or inward-open partially occluded state, limiting our understanding of the conformational changes during transport. To date, it is unclear why both human transporters display different conformational states under the measured conditions, since a recent single molecule FRET study on the bacterial POT DtpA confirmed the inward-open state as the lowest energy state in detergent solution ( 50, 51 ) in agreement with the deposited POT structures. Multiple stabilizing interactions, mainly salt-bridges between the N- and C-terminal bundle, need to be broken and formed during a transport cycle ( 27, 52 ).…”

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

“…They demonstrate that methylation of the phosphatidylethanolamine headgroup tips the conformational equilibrium towards the outward-open state. Such dependence on the chemistry of the membrane was also observed for the transporter DtpA [73]. The authors used the Salipro system to reconstitute the peptide transporter DtpA in soluble discoidal bilayers composed either of phosphatidylethanolamine POPE, phosphatidylserine POPS, phosphatidic acid POPA or brain lipids extract.…”

“…They are also often monomeric or dimeric, which facilitates the labeling strategy, compared to multimeric channels, for example. For these practical reasons, DEER [ 67 , 68 , 69 , 70 ] and smFRET [ 71 , 72 , 73 , 74 ] studies in membrane mimics are heavily biased towards transporters.…”

Spectroscopy of model-membrane liposome-protein systems: complementarity of linear dichroism, circular dichroism, fluorescence and SERS

“…The fluorescence signal of the dansyl labelled liposomes (acceptor) increased in the presence of tryptophan residues of protein MPP1 (donor). Membrane-protein interactions have also been studied by Lasitza-Male et al [19] by probing the dynamics of DtpA protein (a proton-transporter) in various lipid environments (Figure 4). The existence of various lipid fluorophores allows the use of the FRET techique to study lipid layer dynamics.…”