Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Abstract

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di-and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO 2 ]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward-and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO 2 ]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.membrane transporter | proton-dependent oligopeptide transporter | major facilitator superfamily | atomic force microscopy | molecular interactions

Substrate-induced changes in the structural properties of LacY

“…fitted using the worm-like-chain (WLC) model to reveal the contour length (in aa) of the unfolded, stretched LacY polypeptide (25,44). Histograms showing the contour length at which the force peaks reproducibly occurred were generated ( Fig.…”

“…Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26-35).…”

“…Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

Observing a Lipid-Dependent Alteration in Single Lactose Permeases

“…In addition, most force peaks detected for the class 2 FD curves show a much wider contour length distribution compared to the force peaks detected for the class 1 FD curves (Figure 2 and 3). This increase in width suggests greater conformational variability and flexibility of the structural segments stabilizing the inverted LacY topology and possibly indicates the coexistence of multiple intermediate states (Bippes et al, 2013). The suggestion is consistent with the finding of Vitrac et al (Vitrac et al, 2013) that different structural elements of the N-terminal bundle in LacY behave asynchronously in response to varying the PE concentration of the proteoliposomes.…”

“…SMFS is applied to membrane proteins embedded in native or synthetic lipid membranes under physiologic conditions and has been useful for characterizing the stability of secondary structure segments of membrane proteins under a wide range of conditions (Bippes et al, 2009; Bippes et al, 2013; Ge et al, 2011; Janovjak et al, 2003; Kedrov et al, 2005; Park et al, 2007; Sapra et al, 2008; Sapra et al, 2006; Serdiuk et al, 2014; Zocher et al, 2012a; Zocher et al, 2012b). Thus, SMFS is particularly well suited to study conformational states of membrane proteins and the changes they undergo.…”