Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters

Abstract

Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referred to as E1XXE2R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y, or Q1XXE2Y were able to accumulate peptide, whereas those with E1XXQ2R, Q1XXE2R, or Q1XXQ2Y were unable to accumulate peptide, and Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E2 to R-E1, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E1XXE2R and E1XXQ2R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E2 has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.

Keywords: membrane transport; molecular dynamics; peptide transport; protein structure; site-directed mutagenesis.

FIGURE 1.

Overall architecture and conformations of POTs. A, crystal structure of the inward open PepT_So2·Ala-Ala-Ala complex (Protein Data Bank code 4TPJ) (42). The N- and C-terminal domains are colored white and blue, respectively. Side chains shown as sticks correspond to the E₁XXE₂RFXYY motif (green) and the FYING motif (wheat). The substrate is represented as yellow sticks. B, schematic representation of possible conformational changes in POTs during proton/peptide translocation; the proton is represented as a black sphere with spikes, and the peptide is represented by a black rhombus. C, multiple sequence alignment of E₁XXE₂RFXYY and FYXXINXG motifs using mafft from GkPOT (Q5KYD1), YjdL (P39276), YdgR (P77304), YbgH (P75742), hPepT1 (P46059), PepT_So (Q8EHE6), PepT_St (Q5M4H8), and NRT1.1 (Q05085).

FIGURE 2.

A, fluorescence quenching of β-Ala-Lys(AMCA). 0.5 mm β-Ala-Lys(AMCA) was incubated with cells harboring YjdL-WT (A₆₀₀ of 10 for 30 min). The cells were then centrifuged and washed using ice-cold uptake buffer. The fluorescence was measured before and after sonication on a Safire 2 fluorometer at an excitation wavelength and emission wavelength of 340 and 460 nm, respectively. The data shown were normalized to the fluorescence values shown by β-Ala-Lys(AMCA) in intact cells. Error bars indicate S.E. (n = 3). B, Western blots of the motif mutants (n = 3). Only a representative blot is shown.

FIGURE 3.

Accumulation profiles of motif variants Q₁XXE₂Y (A), E₁XXQ₂Y (B), E₁XXE₂Y (C), Q₁XXQ₂Y (D), E₁XXE₂R (E), E₁XXQ₂R (F), Q₁XXE₂R (G), and Q₁XXQ₂R (H). The ratio of substrate accumulated inside the cell with respect to the substrate concentration outside the cell was plotted as a function of time. The assay was performed at pH_o 5.5 (open squares), 6.5 (open circles), and 7.5 (open triangles). The closed symbols represent empty vector. The error bars indicate the S.E. (n = 3).

FIGURE 4.

Molecular dynamics simulations probing the effect of E₂ to Q change. The shortest side chain N–O distances (Å) from Arg³⁶ to Glu³² (gray) and Glu³⁵ (black), respectively, as a function of time in GkPOT-WT (E₁XXE₂R) (A) and E35Q-GkPOT (E₁XXQ₂R) (B) are shown. Insets represent the position of arginine at 0 and 35 ns.

FIGURE 5.

Movement of Helix V as a consequence of salt bridge swapping. A, the movement in Helix V of E35Q-GkPOT accompanying the shift of Arg³⁶ on Helix I. The helices of E35Q-GkPOT at 35 ns (wheat) were aligned to GkPOT-WT at 35 ns (white). B, the root mean square deviation (RMSD) (Å) of the Cα atom of Tyr¹⁶² on Helix V plotted as a function of time in GkPOT-WT (gray) and E35Q-GkPOT (black).

FIGURE 6.

Schematic representation of salt bridge swapping in the E₁XXE₂R motif shown in GkPOT corresponding to the presence of substrate Ala-Ala and proton (red sphere). A, absence of both substrate and proton. B, presence of Ala-Ala alone. C, presence of both substrate and proton.

FIGURE 7.

A, hydrogen bonding network in the PepT_St·AF complex (Protein Data Bank code 4D2C) (17) between the E₁XXE₂R residues in Helix V and Lys¹²⁶ in PepT_St (wheat) and the dipeptide AF (white). The amino acid residue numbering corresponds to PepT_St followed by GkPOT and YjdL. B, superposition of Helices I and V from the 35-ns E35Q-GkPOT (E₁XXQ₂R) structure in green and NRT1.1 protomer B (Protein Data Bank code 4OH3) (43) in wheat, respectively. Arginine is shown as sticks. C, accumulation profiles of V159G-YdgR (open squares), G146V-YjdL (open circles), YjdL-WT (open rhombuses), and YdgR-WT (closed rhombuses). The ratio of substrate accumulated inside the cell with respect to the substrate concentration outside the cell was plotted as a function of time. The assay was performed at pH_o 6.5. The open downward facing triangles represent empty vector. Error bars indicate S.E. (n = 3).

FIGURE 8.

Further destabilization of the binding site as a consequence of E₂ protonation. A, Helix I of E35Q-GkPOT after 35 ns (wheat) superimposed with Helix I of the GkPOT-WT after 35 ns (gray) indicating the change in conformation of Tyr⁴⁰. B, crystal structure of GkPOT displaying the salt bridge between N- (wheat) and C (blue)-domains, i.e. between residues Arg⁴³ and Glu³¹⁰ (yellow). C, the shortest side chain N–O distance (Å) between Arg⁴³ and Glu³¹⁰ as a function of time in GkPOT-WT (black) and E35Q-GkPOT (gray).