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

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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 (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.

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Acknowledgements

This work was funded by the BBSRC Membrane Protein Structure Initiative (MPSI, BBS/B/14418), the EU European Drug Initiative on Channels and Transporters consortium (EDICT, contract 201294), the EPSRC (EP/G035695/1), Invitrogen Corporation and the University of Leeds. The authors thank Malcolm Levitt (University of Southampton) for support. MR thanks the Islamic Development Bank (IDB) and the University of Leeds for a PhD studentship and Mike McPherson (University of Leeds) for advice. Mass spectrometry was performed using the Biomolecular Mass Spectrometry Facility in the Astbury Centre for Structural Molecular Biology with assistance from Alison Ashcroft (University of Leeds). The authors also thank Denise Ashworth and Jeff Keen (University of Leeds) for performing DNA and protein sequencing.

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  1. School of BioMedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

    Moazur Rahman, Fouzia Ismat, Li Jiao, Jocelyn M. Baldwin, David J. Sharples, Stephen A. Baldwin & Simon G. Patching

  2. Drug Discovery and Structural Biology Group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

    Moazur Rahman & Fouzia Ismat

  3. Faculty of Biological Sciences, University of Leeds, Astbury Building, Leeds, LS2 9JT, UK

    Simon G. Patching

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  1. Moazur Rahman
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Correspondence to Simon G. Patching.

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This paper is dedicated to the memory of Stephen A. Baldwin.

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Rahman, M., Ismat, F., Jiao, L. et al. Characterisation of the DAACS Family Escherichia coli Glutamate/Aspartate-Proton Symporter GltP Using Computational, Chemical, Biochemical and Biophysical Methods. J Membrane Biol 250, 145–162 (2017). https://doi.org/10.1007/s00232-016-9942-x

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