TY - JOUR

T1 - Salt-bridge Swapping in the EXXERFXYY Motif of Proton Coupled Oligopeptide Transporters

AU - Aduri, Nanda G

AU - Prabhala, Bala K

AU - Ernst, Heidi A

AU - Jørgensen, Flemming S

AU - Olsen, Lars

AU - Mirza, Osman

N1 - Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

PY - 2015

Y1 - 2015

N2 - Proton-coupled oligopeptide transporters (POTs) couple the inwards transport of di- or tripeptides with an inwards-directed transport of protons. Evidence from several studies of different POTs have pointed towards 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 E. coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y or Q1XXE2Y, were able to accumulate peptide, whereas E1XXQ2R, Q1XXE2R or Q1XXQ2Y, were unable to accumulate; Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intra-motif 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.

AB - Proton-coupled oligopeptide transporters (POTs) couple the inwards transport of di- or tripeptides with an inwards-directed transport of protons. Evidence from several studies of different POTs have pointed towards 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 E. coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y or Q1XXE2Y, were able to accumulate peptide, whereas E1XXQ2R, Q1XXE2R or Q1XXQ2Y, were unable to accumulate; Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intra-motif 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.

U2 - 10.1074/jbc.M115.675603

DO - 10.1074/jbc.M115.675603

M3 - Journal article

C2 - 26483552

VL - 290

SP - 29931

EP - 29940

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -