title: Characterisation of the DAACS Family Escherichia coli Glutamate/Aspartate-Proton Symporter GltP Using Computational, Chemical, Biochemical and Biophysical Methods.
abstract: 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 (Km) for3H-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.
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.|Drug Discovery and Structural Biology Group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
authors.forename: Moazur
authors.initials: M
authors.lastname: Rahman
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.|Drug Discovery and Structural Biology Group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
authors.forename: Fouzia
authors.initials: F
authors.lastname: Ismat
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Li
authors.initials: L
authors.lastname: Jiao
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Jocelyn M
authors.initials: JM
authors.lastname: Baldwin
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sharples
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.affiliation: School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK. s.g.patching@leeds.ac.uk.|Faculty of Biological Sciences, University of Leeds, Astbury Building, Leeds, LS2 9JT, UK. s.g.patching@leeds.ac.uk.
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D013050:Spectrometry, Fluorescence
chemical_list: D027322:Amino Acid Transport System X-AG; D018698:Glutamic Acid
keywords: Fluorescence spectroscopy; Glutamate transport; Homology modelling; Phylogenetic analysis; Site-directed mutagenesis; Solid-state NMR