Biomedical subjects

Eric Gouaux

Publications and source records attributed to Eric Gouaux.

At least 19 recordsLinked to original sources

PubMed2007

Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.

Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

Amino Acid Motifs↗

PubMed2006

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor.

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.

Animals↗

PubMed2006

Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.

Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.

Animals↗

PubMed2005

Principles of selective ion transport in channels and pumps.

The transport of ions across the membranes of cells and organelles is a prerequisite for many of life's processes. Transport often involves very precise selectivity for specific ions. Recently, atomic-resolution structures have been determined for channels or pumps that are selective for sodium, potassium, calcium, and chloride: four of the most abundant ions in biology. From these structures we can begin to understand the principles of selective ion transport in terms of the architecture and detailed chemistry of the ion conduction pathways.

Biological Transport, Active↗

PubMed2005

Subunit arrangement and function in NMDA receptors.

Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

Animals↗

PubMed2005

Mechanism of positive allosteric modulators acting on AMPA receptors.

Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the "hinge" in the ligand-binding core "clamshell" that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

Allosteric Regulation↗

PubMed2005

Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore.

Pathogenic Vibrio cholerae secrete V. cholerae cytolysin (VCC), an 80 kDa pro-toxin that assembles into an oligomeric pore on target cell membranes following proteolytic cleavage and interaction with cell surface receptors. To gain insight into the activation and targeting activities of VCC, we solved the crystal structure of the pro-toxin at 2.3A by X-ray diffraction. The core cytolytic domain of VCC shares a fold similar to the staphylococcal pore-forming toxins, but in VCC an amino-terminal pro-domain and two carboxy-terminal lectin domains decorate the cytolytic domain. The pro-domain masks a protomer surface that likely participates in inter-protomer interactions in the cytolytic oligomer, thereby explaining why proteolytic cleavage and movement of the pro-domain is necessary for toxin activation. A single beta-octyl glucoside molecule outlines a possible receptor binding site on one lectin domain, and removal of this domain leads to a tenfold decrease in lytic activity toward rabbit erythrocytes. VCC activated by proteolytic cleavage assembles into an oligomeric species upon addition of soybean asolectin/cholesterol liposomes and this oligomer was purified in detergent micelles. Analytical ultracentrifugation and crystallographic analysis indicate that the resulting VCC oligomer is a heptamer. Taken together, these studies define the architecture of a pore forming toxin and associated lectin domains, confirm the stoichiometry of the assembled oligomer as heptameric, and suggest a common mechanism of assembly for staphylococcal and Vibrio cytolytic toxins.

Animals↗

PubMed2005

Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.

Amino Acid Sequence↗

PubMed2005

Mechanism of partial agonist action at the NR1 subunit of NMDA receptors.

Partial agonists produce submaximal activation of ligand-gated ion channels. To address the question of partial agonist action at the NR1 subunit of the NMDA receptor, we performed crystallographic and electrophysiological studies with 1-aminocyclopropane-1-carboxylic acid (ACPC), 1-aminocyclobutane-1-carboxylic acid (ACBC), and 1-aminocyclopentane-1-carboxylic acid (cycloleucine), three compounds with incrementally larger carbocyclic rings. Whereas ACPC and ACBC partially activate the NMDA receptor by 80% and 42%, respectively, their cocrystal structures of the NR1 ligand binding core show the same degree of domain closure as found in the complex with glycine, a full agonist, illustrating that the NR1 subunit provides a new paradigm for partial agonist action that is distinct from that of the evolutionarily related GluR2, AMPA-sensitive receptor. Cycloleucine behaves as an antagonist and stabilizes an open-cleft conformation. The NR1-cycloleucine complex forms a dimer that is similar to the GluR2 dimer, thereby suggesting a conserved mode of subunit-subunit interaction in AMPA and NMDA receptors.

Amino Acids, Cyclic↗

PubMed2004

Structure of a glutamate transporter homologue from Pyrococcus horikoshii.

Glutamate transporters are integral membrane proteins that catalyse the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.

Amino Acid Sequence↗

PubMed2004

High resolution crystallographic studies of alpha-hemolysin-phospholipid complexes define heptamer-lipid head group interactions: implication for understanding protein-lipid interactions.

The alpha-hemolysin is an archetypal pore-forming protein that is secreted from Staphylococcus aureus as a water-soluble monomer. When the monomer binds to the membrane of a susceptible cell, the membrane-bound molecules assemble into the lytic heptamer. Although a bilayer or a bilayer-like environment are essential to toxin assembly, there is no high resolution information on toxin-phospholipid complexes. We have determined the structures of detergent-solubilized alpha-hemolysin heptamer bound to glycerophosphocholine or dipropanoyl glycerophosphocholine at 1.75-1.80 A resolution and 110 K. The phosphocholine head group binds to each subunit in a crevice between the rim and the stem domains. The quaternary ammonium group interacts primarily with aromatic residues, whereas the phosphodiester moiety interacts with a conserved arginine residue. These structures provide a molecular basis for understanding why alpha-hemolysin preferentially assembles on membranes comprised of phosphocholine lipids.

Bacterial Proteins↗

PubMed2003

Structure and function of AMPA receptors.

The application of X-ray diffraction has allowed the structure of the ligand-binding core of AMPA receptors to be determined. Here I review the insights that this has given into the molecular mechanisms of activation and desensitization of these receptors.

Animals↗

PubMed2003

Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus.

Catalysis of glutamate transport across cell membranes and coupling of the concentrative transport to sodium, proton, and potassium gradients are processes fundamental to organisms in all kingdoms of life. In bacteria, glutamate transporters participate in nutrient uptake, while in eukaryotic organisms, the transporters clear glutamate from the synaptic cleft. Even though glutamate transporters are crucial to the viability of many life forms, little is known about their structure and quaternary organization. In particular, the subunit stoichiometry of these polytopic integral membrane proteins has not been unequivocally defined. Determination of the native molecular mass of membrane proteins is complicated by their lability in detergent micelles and by their association with detergent and/or lipid molecules. Here we report the purification of glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus in a monodisperse, detergent-solubilized state. Characterization of both transporters either by chemical cross-linking and mass spectrometry or by size-exclusion chromatography and in-line laser light scattering, refractive index, and ultraviolet absorption measurements shows that the transporters have a trimeric quaternary structure. Limited proteolysis further defines regions of primary structure that are exposed to aqueous solution. Together, our results define the subunit stoichiometry of high-affinity glutamate transporters from B. caldotenax and B. stearothermophilus and localize exposed and accessible elements of primary structure. Because of the close amino acid sequence relationship between bacterial and eukaryotic transporters, our results are germane to prokaryotic and eukaryotic glutamate and neutral amino acid transporters.

Amino Acid Sequence↗

PubMed2003

Structural basis for modulation and agonist specificity of HCN pacemaker channels.

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.

Amino Acid Sequence↗

PubMed2003

Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core.

Excitatory neurotransmission mediated by the N-methyl-D-aspartate subtype of ionotropic glutamate receptors is fundamental to the development and function of the mammalian central nervous system. NMDA receptors require both glycine and glutamate for activation with NR1 and NR2 forming glycine and glutamate sites, respectively. Mechanisms to describe agonist and antagonist binding, and activation and desensitization of NMDA receptors have been hampered by the lack of high-resolution structures. Here, we describe the cocrystal structures of the NR1 S1S2 ligand-binding core with the agonists glycine and D-serine (DS), the partial agonist D-cycloserine (DCS) and the antagonist 5,7-dichlorokynurenic acid (DCKA). The cleft of the S1S2 'clamshell' is open in the presence of the antagonist DCKA and closed in the glycine, DS and DCS complexes. In addition, the NR1 S1S2 structure reveals the fold and interactions of loop 1, a cysteine-rich region implicated in intersubunit allostery.

Amino Acid Sequence↗

PubMed2003

Probing the function, conformational plasticity, and dimer-dimer contacts of the GluR2 ligand-binding core: studies of 5-substituted willardiines and GluR2 S1S2 in the crystal.

Numerous naturally occurring and synthetic alpha-amino acids act as agonists on (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptors but nevertheless display significant differences in their functional properties and modes of interaction. The 5-substituted willardiines are a series of compounds that exhibit a range of affinities, act as partial agonists, and give rise to intermediate levels of activation and desensitization. However, the molecular basis for the activities of 5-substituted willardiines has not been conclusively elaborated at the level of atomic resolution. Here we provide insight into the molecular basis of the potency and efficacy elicited by the 5-substituted willardiines on the basis of cocrystal structures with the GluR2 ligand-binding core. We also show that the crystallized ligand-binding core has an affinity for agonists similar to the ligand-binding core in solution. Analysis of multiple crystal lattices suggests modes by which the ligand-binding core dimers interact in the tetrameric receptor. These studies further our understanding of how subtle differences in the structures of agonists are correlated to changes in the conformation of residues and water molecules in the immediate binding pocket and to the degree of domain closure.

Alanine↗

PubMed2003

Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes.

The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical techniques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully closed conformation in the AMPA-bound form. Our results indicate that agonists can induce a range of conformations in the GluR2 ligand-binding core and that domain closure is directly correlated to channel activation. The partially closed, AMPA-bound conformation of the L650T mutant likely captures the structure of an agonist-bound, inactive state of the receptor. Together with previously solved structures, we have determined a mechanism of agonist binding and subsequent conformational rearrangements.

Amino Acid Substitution↗

PubMed2003

Three-dimensional structure of the ligand-binding core of GluR2 in complex with the agonist (S)-ATPA: implications for receptor subunit selectivity.

Two X-ray structures of the GluR2 ligand-binding core in complex with (S)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid ((S)-ATPA) have been determined with and without Zn(2+) ions. (S)-ATPA induces a domain closure of ca. 21 degrees compared to the apo form. The tert-butyl moiety of (S)-ATPA is buried in a partially hydrophobic pocket and forces the ligand into the glutamate-like binding mode. The structures provide new insight into the molecular basis of agonist selectivity between AMPA and kainate receptors.

Binding Sites↗