Salt-bridge Dynamics Control Substrate-induced Conformational Change in the Membrane Transporter GlpT

Abstract

SummaryActive transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P i ) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inwardfacing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter-and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate/phosphate antiporters.

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

“…Because even conservative mutations distant from the substrate translocation pathway, such as L190V or L324V, lead to changes in the apparent affinity for selected substrates, we propose that YdgR undergoes large and dynamic conformational transitions, as has been shown for LacY and GlpT (27,30). Due to the large number of mutations we isolated in helix 7, it seems that in particular the interhelical interface of this helix is critical for proper protein function.…”

“…Several polar or charged residues that were isolated as LOF mutants (Glu 56 , Ser 59 , Asn 300 , Arg 305 , Asn 306 ) are located in the periplasmic halves of helices 2 and 7, and may form part of a hydrogen bonding network critical for closing of the periplasmic cavity, as seen in LacY and GlpT (27,28). As the putative gating residues of the peptide transporter PepT so , His 61 and Glu…”

Cotranslational folding of membrane proteins probed by arrest-peptide–mediated force measurements

“…In the crystal structure of GlpT, E299 in the C-terminal TMH8 is located close to possible interaction partners K46 and H165 (30), both of which are buried deep in the protein interior (Fig. 3A).…”

Shared Molecular Mechanisms of Membrane Transporters

“…What is the nature of the energetic barrier that is lowered by substrate binding? After the crystal structures of LacY and GlpT were determined, it was proposed that this barrier involves the breakage and re-formation of salt bridges that hold the N-and C-terminal bundles together (91,92). Indeed, interbundle salt bridges have consistently been found in MFS transporters and are often proximal to the central cavity (5,63,64,73,93).…”