Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

Engineering the glutamate transporter homologue GltPh using protein semisynthesis.

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 homologue of glutamate transporters. Semisynthesis allows 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 analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of 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.

Glutamate transporters in the biology of malignant gliomas.

Malignant gliomas are relentless tumors that offer a dismal clinical prognosis. They develop many biological advantages that allow them to grow and survive in the unique environment of the brain. The glutamate transporters system x c (-) and excitatory amino acid transporters (EAAT) are emerging as key players in the biology and malignancy of these tumors. Gliomas manipulate glutamate transporter expression and function to alter glutamate homeostasis in the brain, which supports their own growth, invasion, and survival. As a consequence, malignant cells are able to quickly destroy and invade surrounding normal brain. Recent findings are painting a larger picture of these transporters in glioma biology, and as such are providing opportunities for clinical intervention for patients. This review will detail the current understanding of glutamate transporters in the biology of malignant gliomas and highlight some of the unique aspects of these tumors that make them so devastating and difficult to treat.

Induced fit substrate binding to an archeal glutamate transporter homologue.

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles. At low millimolar [Na(+)], the addition of L-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na(+)], the kinetics were dominated by a fast component [k(obs,fast); K(D) (Na(+)) = 22 ± 3 mM, n(Hill )= 1.7 ± 0.3] with values of k(obs,fast) rising in a saturable manner to ≈ 500 s(-1) (at 6 °C) with increasing [L-aspartate]. The binding kinetics of L-aspartate differed from the binding kinetics of two alternative substrates: L-cysteine sulfinic acid and d-aspartate. L-cysteine sulfinic acid bound with higher affinity than L-aspartate but involved lower saturating rates, whereas the saturating rates after D-aspartate binding were higher. Thus, after the association of two Na(+) to the empty transporter, GltPh binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for GltPh than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.

Cysteine scanning mutagenesis of transmembrane helix 3 of a brain glutamate transporter reveals two conformationally sensitive positions.

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3' site, and another, Tyr-124, appears close to both Na3' and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3' and Na1 underlies the protection by sodium.

Large collective motions regulate the functional properties of glutamate transporter trimers.

Glutamate transporters clear synaptically released glutamate to maintain precise communication between neurons and limit glutamate neurotoxicity. Although much progress has been made on the topology, structure, and function of these carriers, few studies have addressed large-scale structural motions collectively associated with substrate transport. Here we show that a series of single cysteine substitutions in the helical hairpin HP2 of excitatory amino acid transporter 1 form intersubunit disulfide cross-links within the trimer. After cross-linking, substrate uptake, but not substrate-activated anion conductance, is completely inhibited in these mutants. These disulfide bridges link residue pairs > 40 Å apart in the outward-facing crystal structure, and can be explained by concerted subunit movements predicted by the anisotropic network model (ANM). The existence of these global motions is further supported by the observation that single cysteine substitutions at the extracellular part of the transmembrane domain 8 can also be cross-linked by copper phenanthroline as predicted by the ANM. Interestingly, the transport domain in the un-cross-linked subunit of the trimer assumes an inward-facing orientation, suggesting that individual subunits potentially undergo separate transitions between outward- and inward-facing forms, rather than an all-or-none transition of the three subunits, a mechanism also supported by ANM-predicted intrinsic dynamics. These results shed light on how large collective motions contribute to the functional dynamics of glutamate transporters.

The 3-4 loop of an archaeal glutamate transporter homolog experiences ligand-induced structural changes and is essential for transport.

Glutamatergic synaptic transmission is terminated by members of the excitatory amino acid transporter (EAAT) family of proteins that remove glutamate from the synaptic cleft by transporting it into surrounding glial cells. Recent structures of a bacterial homolog suggest that major motions within the transmembrane domain translocate the substrate across the membrane. However, the events leading to this large structural rearrangement are much less clear. Two reentrant loops have been proposed to act as extracellular and intracellular gates, but whether other regions of these proteins play a role in the transport process is unknown. We hypothesized that transport-related conformational changes could change the solvent accessibilities of affected residues, as reflected in protease sensitivity or small-molecule reactivity. In the model system Glt(Ph), an archaeal EAAT homologue from Pyrococcus horikoshii, limited trypsin proteolysis experiments initially identified a site in the long extracellular loop that stretches between helices 3 and 4 that becomes protected from proteolysis in the presence of a substrate, L-aspartate, or an inhibitor, DL-TBOA in the presence of Na(+), the cotransported ion. Using a combination of site-directed cysteine-scanning mutagenesis and fluorescein-5-maleimide labeling we found that positions throughout the loop experience these ligand-induced conformational changes. By selectively cleaving the 3-4 loop (via introduced Factor Xa sites) we demonstrate that it plays a vital role in the transport process; though structurally intact, the cleaved proteins are unable to transport aspartate. These results inculcate the 3-4 loop as an important player in the transport process, a finding not predicted by any of the available crystal structures of Glt(Ph).

Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats.

Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 A apart in the crystal structures of GltPh, but approximately = 10 A apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)(3) abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-beta-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.

Transport mechanism of a bacterial homologue of glutamate transporters.

Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, Glt(Ph), which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt(Ph) in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt(Ph) and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.

In situ monitoring of the orientated assembly of strep-tagged membrane proteins on the gold surface by surface enhanced infrared absorption spectroscopy.

Surface enhanced infrared absorption spectroscopy (SEIRAS) has been employed to monitor the orientated assembly of a strep-tagged membrane protein on the gold surface via a streptavidin/biotin interlayer. The high surface sensitivity of SEIRAS allows for tracking the individual assembling steps on the molecular level. The sequence of surface modification steps comprises: (i) cross-linking of biotin to the self-assembled monolayer of cysteamine along the gold surface; (ii) adsorption of streptavidin to and desorption from the biotin layer; and (iii) adsorption of the strep-tagged membrane protein ecgltP (glutamate transporter of E. coli) on the streptavidin/biotin layer. The analysis of the SEIRA spectra reveals that the biotin layer undergoes a phase transition from an isotropic orientation to a densely packed layer during coupling to the cysteamine monolayer. Formation of the densely packed layer weakens the interaction between streptavidin and the biotin layer but yields a binding specificity of 80%. The specificity of strep-tagged ecgltP to the streptavidin layer is with 60% only modest. Nevertheless, the streptavidin/biotin interlayer reveals a higher regeneration propensity than the His-tag/Ni-NTA interlayer.

Voltage-independent sodium-binding events reported by the 4B-4C loop in the human glutamate transporter excitatory amino acid transporter 3.

Glutamate transporters (excitatory amino acid transporter (EAATs)) are critical for normal excitatory signaling and maintaining subtoxic glutamate concentrations in mammalian central nervous system. Recently, a crystal structure for a homologous transporter in bacteria was reported. Still, little is understood regarding the mechanism of substrate uptake. In transmembrane domain 4, the mammalian EAATs contain a stretch of over 50 amino acids (4B-4C loop) that are absent in the bacterial protein. These residues have been suggested to be located in the large extracellular vestibule seen in the crystal structure. State-dependent trypsin-cleavage sites have been reported in this region, suggesting that the 4B-4C loop undergoes significant conformational changes. Here we employed substituted cysteine accessibility, voltage clamp fluorometry, and fluorescence resonance energy transfer on oocytes expressing mutant EAAT3 transporters to determine the location and functionality of the 4B-4C loop. We find that this loop extends from near the center of the protein and that the majority of the residues are positioned on the outer perimeter of the protein, rather than inside the vestibule. Our fluorescence resonance energy transfer measurements demonstrated that these residues do not undergo large scale motions during glutamate uptake. However, our voltage clamp fluorometry studies indicate that these residues report on Na(+) and glutamate binding-induced conformational changes, including a previously un-described voltage-independent component of Na(+) binding to the transporter. The finding that residues far from the glutamate-binding site report on several different types of binding events suggests that the series of small conformational changes that accomplish glutamate uptake extend throughout the transporter structure.

Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters.

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent glutamate concentrations from reaching neurotoxic levels. Glutamate transporters play an important role in ischemic neuronal death during stroke and have been implicated in epilepsy and amytropic lateral sclerosis. However, the molecular structure and the glutamate-uptake mechanism of these transporters are not well understood. The most recent models of glutamate transporters have three or five subunits, each with eight transmembrane domains, and one or two membrane-inserted loops. Here, using fluorescence resonance energy transfer (FRET) analysis, we have determined the relative position of the extracellular regions of these domains. Our results are consistent with a trimeric glutamate transporter with a large (>45 A) extracellular vestibule. In contrast to other transport proteins, our FRET measurements indicate that there are no large-scale motions in glutamate transporters and that glutamate uptake is accompanied by relatively small motions around the glutamate-binding sites. The large extracellular vestibule and the small-scale conformational changes could contribute to the fast kinetics predicted for glutamate transporters. Furthermore, we show that, despite the multimeric nature of glutamate transporters, the subunits function independently.

Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.