Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

Abstract

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.

Keywords: X-ray crystallography; membrane transporter; molecular dynamics simulation.

Fig. 1.

High-resolution crystal structure of GkPOT. (A and B) Crystal structure of GkPOT in the substrate-free form, viewed parallel to the membrane (A) and from the intracellular side (B). The N- (H1–H6) and C-terminal (H7–H12) bundles and the additional helices (HA and HB) are colored cyan, pink, and yellow, respectively. Sulfate ions, waters, and lipid molecules are shown in ball-and-stick representations. The rectangles indicate the peptide binding site.

Fig. 2.

Functional analysis of GkPOT by a liposome-based assay. (A) Competition assay with ³H-labeled (Ala)₂ peptide. “No competitor” represents the conditions in which only the ³H di-alanine peptide was present in the external buffer, without additional unlabeled peptide. Error bars indicate the SDs from triplicate experiments. Asterisks indicate that activity was not detectable. (B) Effect of substitutions within the peptide binding site on H⁺-driven uptake. “CCCP” refers to the addition of the proton ionophore, carbonyl cyanide m-chlorophenyl hydrazine, to the external buffer. (C) Effect of equivalent substitutions on peptide-driven counterflow uptake.

Fig. 3.

Alafosfalin-bound crystal structure of the GkPOT E310Q variant. (A and B) Crystal structure of the GkPOT E310Q variant in complex with alafosfalin, viewed parallel to the membrane (A) and from the intracellular side (B). The regions within rectangles in Fig. 1 A and B roughly correspond to the expanded regions in A and B, respectively.

Fig. 4.

Structural transitions revealed by MD simulations. (A and B) Time series of the distances between the cytosolic-side helices of the N- and C-terminal bundles (residues 141–156 and 420–440, respectively) in the S-E310 and S-E310p-FF simulations. The arrows indicate the most closed frames. (C–E) Cylinder representations of the initial structure (C) and the most closed snapshots in the S-E310 (D) and S-E310p-FF (E) simulations. The color code for the domains is the same as in Fig. 1. The arrows in C indicate the distances measured in A and B.

Fig. 5.

Proposed model for the substrate/H⁺-coupled structural transition between the inward-open and partially occluded states. Half of the symport cycle, involving the inward-open and occluded forms, is shown. The transitions involved in the physiological symport cycle are indicated with black arrows; others are shown with gray arrows. The states captured in the present crystal structures are enclosed by rectangles. The details of the model are discussed in the main text.