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 (K) for H-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.

The high‐energy transition state of the glutamate transporter homologue GltPh

“…This property is in line with observations that enzymes from thermophilic organisms, which evolved to be stable and functional at high temperatures, show low activity at ambient temperature (Somero, 2004; Elias, Wieczorek et al , 2014). Consistently, glutamate transporters from mesophilic bacteria are more active than Glt Ph (Tolner, Ubbink‐Kok et al , 1995; Gaillard, Slotboom et al , 1996; Yernool, Boudker et al , 2003; Rahman, Ismat et al , 2017). Also, structurally very similar human EAATs (Canul‐Tec et al , 2017) are ~20 to 10,000‐fold faster (Vandenberg & Ryan, 2013).…”

Linking function to global and local dynamics in an elevator-type transporter

“…Furthermore, a comparison of enzymes from thermophilic and mesophilic organisms showed that increased activity at lower temperatures correlates with increased protein flexibility ( 59 ). Glt Ph originates from a hyperthermophilic archaeon and shows much lower activity at room temperature than its mesophilic counterparts ( 23 , 38 , 60 – 63 ). Therefore, it is, perhaps, not unexpected that the protein demonstrates rigidity at ambient temperatures in HDX-MS experiments, and mutations leading to increased protein flexibility also lead to increased activity, akin to natural thermal adaptations.…”

DcuA of aerobically grown Escherichia coli serves as a nitrogen shuttle (L‐aspartate/fumarate) for nitrogen uptake

“…DcuA is responsible for the uptake of L‐aspartate as a nitrogen source during aerobic growth of E. coli . Remarkably, DctA, DauA, GltP, DcuB and DcuC, which are known to catalyze L‐aspartate transport (Kay and Kornberg, ; Wallace et al ., ; Raunser et al ., ; Karinou et al ., ; Rahman et al ., ), were not used for this function. The nearly complete loss of growth on L‐aspartate in the dcuA mutant (Table ) shows also that the ABC glutamate/aspartate transporter GltIJKL (Willis and Furlong ) has no significant role under these conditions.…”

“…Under acidic conditions (pH 5), transporter DauA, a member of the solute carrier SCL26 and sulfate (SulP) transporter family, takes over the function of DctA (Karinou et al ., ). Transporter GltP, which catalyzes the uptake of glutamate, has low side activity for L‐aspartate (Raunser et al ., ; Rahman et al ., ) and may contribute to the uptake of L‐aspartate. In addition, DcuA a member of the DcuA/DcuB C 4 ‐dicarboxylate transporter family is expressed constitutively under aerobic conditions and has been suggested to function in aerobic metabolism (Golby et al ., ).…”