Characterisation of the DAACS Family Escherichia coli Glutamate/Aspartate-Proton Symporter GltP Using Computational, Chemical, Biochemical and Biophysical Methods.

Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh. Topology predictions and comparisons with the crystal structure of GltPh were consistent with eight transmembrane-spanning α-helices and two hairpin re-entrant loops in GltP. Amplified expression of recombinant GltP with C-terminal affinity tags was achieved at 10% of total membrane protein in E. coli and purification to homogeneity with a yield of 0.8 mg/litre. Binding of substrates to GltP in native inner membranes and to purified protein solubilised in detergent was observed and quantified using solid-state NMR and fluorescence spectroscopy, respectively. A homology model of GltP docked with L-glutamate identified a putative binding site and residues predicted to interact with substrate. Sequence alignments identified further highly conserved residues predicted to have essential roles in GltP function. Residues were investigated by measuring transport activities, kinetics and response to thiol-specific reagents in 42 site-specific mutants compared with cysteine-less GltP (C256A) having an apparent affinity of initial rate transport (K m) for 3H-L-glutamate of 22.6 ± 5.5 µM in energised E. coli cells. This confirmed GltP residues involved in substrate binding and transport, especially in transmembrane helices VII and VIII.

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR.

Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D(2)O with D-[(13)C(6)]glucose and (13)C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The (13)C chemical shift values for D-[(13)C(6)]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the ß-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.