Engineering the Glutamate Transporter Homologue Glt_Ph Using Protein Semisynthesis

Abstract

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity and the coupling of substrate and ion transport are not well understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homolog of glutamate transporters. Semisynthesis enables the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site with the unnatural analog, citrulline. Our studies demonstrate that Arg397 is required for high affinity substrate binding and based on our results we propose that Arg397 is involved in a Na+- dependent remodeling of the substrate binding site required for high affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5

“…The latter possibility seems more likely because the geometry of all three Na + sites is preserved, at least in cysteine-bound Glt Ph -R397C, and it is unlikely that some of these sites are unoccupied. In addition, reduced substrate and Na + ion coupling was previously observed when Arg397 was replaced with the unnatural amino-acid citrulline lacking positive charge 52 . An apparent number of ions coupled to binding of L-glutamine and benzylcysteine is further reduced to ~ 1 as would be expected if Na2 site were not used.…”

“…The proposed reduced effects of Na + -induced twisting of the NMDGT motif may explain the reduced Na + -coupling in the ASCTs and Glt Ph -R397C compared with transporters that have an arginine residue at this position. Indeed tighter Na + binding and weaker coupling between substrate and Na + binding has also been observed in a Glt Ph variant where R397 was replaced by a neutral citrulline moiety using chemical protein ligation 52 .…”

The Hill analysis and co-ion–driven transporter kinetics

“…Glt Ph is a member of the DAACS family of transporters that contains members from all domains of life, ranging from transporters for glutamate and neutral amino acid uptake in bacteria to excitatory neurotransmitter transporters in the central nervous system ( Slotboom et al, 1999 ; Focke et al, 2013 ). The crystal structure of Glt Ph prompted many functional studies of the transporter to relate structure to mechanism ( Boudker et al, 2007 ; Ryan and Mindell, 2007 ; Reyes et al, 2009 , 2013 ; Ryan et al, 2009 ; Groeneveld and Slotboom, 2010 ; Akyuz et al, 2013 , 2015 ; Erkens et al, 2013 ; Ewers et al, 2013 ; Hänelt et al, 2013 ; Jensen et al, 2013 ; Mulligan and Mindell, 2013 ; Verdon et al, 2014 ; Focke et al, 2015 ; Machtens et al, 2015 ). For the discussion here, it is relevant that rates of aspartate transport as a function of the co-ion concentration have been measured at fixed aspartate concentrations, and conversely, that apparent affinity constants for aspartate have been measured at fixed co-ion concentrations ( Boudker et al, 2007 ; Reyes et al, 2013 ; Verdon et al, 2014 ).…”

A facile approach for the in vitro assembly of multimeric membrane transport proteins

“…Glt Ph migrates as a monomer on SDS-PAGE, suggesting that SDS dissociates Glt Ph into subunits ( Yernool et al, 2004 ). Glutaraldehyde cross-linking confirmed complete dissociation of the trimeric species by SDS ( Focke et al, 2015 ). While crosslinking of the native Glt Ph gives a protein band on SDS-PAGE corresponding to the trimer, crosslinking following SDS treatment only yields monomers.…”

“…Each subunit of Glt Ph has a complex topology with eight transmembrane and two reentrant hairpin segments. Remarkably, we have previously successfully refolded Glt Ph from a completely unfolded state by using lipid vesicles to obtain an active native-like protein ( Focke et al, 2015 ). Here, we investigated whether Glt Ph can be reassembled in vitro from dissociated subunits using lipid vesicles ( Figure 1B ).…”