Dynamics and allosteric potential of the AMPA receptor N-terminal domain

Glutamate-gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell-shaped segments. The distal, N-terminal domain (NTD) has allosteric potential in NMDA-type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped-open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter-rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development.     

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.     

Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.     

Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit

Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure. Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP. In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit. Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft. Upon Zn binding the two lobes close tightly around the divalent cation. Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand. Such a functional organization may apply to other eukaryotic iGluRs.     

The structure of a mixed GluR2 ligand-binding core dimer in complex with (S)-glutamate and the antagonist (S)-NS1209

Ionotropic glutamate receptors (iGluRs) mediate fast synaptic transmission between cells of the central nervous system and are involved in various aspects of normal brain function. iGluRs are implicated in several brain disorders, e.g. in the high-frequency discharge of impulses during an epileptic seizure. (RS)-NS1209 functions as a competitive antagonist at 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionate receptors, and shows robust preclinical anticonvulsant and neuroprotective effects. This study explores 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionate receptor binding and selectivity of this novel class of antagonists. We present here the first X-ray structure of a mixed GluR2 ligand-binding core dimer, with the high-affinity antagonist (S)-8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,-tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2,3-dione-3-O-(4-hydroxybutyrate-2-yl)oxime [(S)-NS1209] in one protomer and the endogenous ligand (S)-glutamate in the other. (S)-NS1209 stabilises an even more open conformation of the D1 and D2 domains of the ligand-binding core than that of the apo structure due to steric hindrance. This is the first time ligand-induced hyperextension of the binding domains has been observed. (S)-NS1209 adopts a novel binding mode, including hydrogen bonding to Tyr450 and Gly451 of D1. Parts of (S)-NS1209 occupy new areas of the GluR2 ligand-binding cleft, and bind near residues that are not conserved among receptor subtypes. The affinities of (RS)-NS1209 at the GluR2 ligand-binding core as well as at GluR1-6 and mutated GluR1 and GluR3 receptors have been measured. Two distinct binding affinities were observed at the GluR3 and GluR4 receptors. In a functional in vitro assay, no difference in potency was observed between GluR2(Q)(o) and GluR3(o) receptors. The thermodynamics of binding of the antagonists (S)-NS1209, DNQX and (S)-ATPO to the GluR2 ligand-binding core have been determined by displacement isothermal titration calorimetry. The displacement of (S)-glutamate by all antagonists was shown to be driven by enthalpy.     

Molecular modeling of the GM-CSF and IL-3 receptor complexes.

A model for the structure of the cytokine interleukin-3 (IL-3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin-5 and granulocyte macrophage colony stimulating factor (GM-CSF). In addition, models of the receptor complexes of GM-CSF and IL-3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM-CSF and IL-3 to their receptors.     

Structure of the zinc‐bound amino‐terminal domain of the NMDA receptor NR2B subunit

N‐methyl‐D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.     

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.     

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.     

Structural biology of MCM helicases

The eukaryotic MCM2-7 complex is recruited onto origins of replication during the G1 phase of the cell cycle and acts as the main helicase at the replication fork during the S phase. Over the last few years a number of structural reports on MCM proteins using both electron microscopy and protein crystallography have been published. The crystal structures of two (almost) full-length archaeal homologs provide the first atomic pictures of a MCM helicase. However one of the structures is at low resolution and the other is of an inactive MCM. Moreover, both proteins are monomeric in the crystal, whereas the activity of the complex is critically dependent on oligomerization. Lower resolution structures derived from electron microscopy studies are therefore crucial to complement the crystallographic analysis and to assemble the multimeric complex that is active in the cell. A critical analysis of all the structural results elucidates the potential conformational changes and dynamic behavior of MCM helicase to provide a first insight into the gamut of molecular configurations adopted during the processes of DNA melting and unwinding.     

Structural insights into the substrate specificity and activity of ervatamins, the papain-like cysteine proteases from a tropical plant, Ervatamia coronaria

Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin-A, ervatamin-B and ervatamin-C are three such papain-like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin-A and ervatamin-C, complexed with an irreversible inhibitor 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane (E-64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (k(cat)/K(m)) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin-C, in the S2 pocket of ervatamin-A (Ala67-->Tyr) results in a 57-fold increase in its k(cat)/K(m) value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin-A, which has been subsequently explained at the molecular level from the three-dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity.     

Emerging models of glutamate receptor ion channel structure and function

Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6?? resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Modeling, docking, and simulation of the major facilitator superfamily

X-ray structures are known for three members of the Major Facilitator Superfamily (MFS) of membrane transporter proteins, thus enabling the use of homology modeling to extrapolate to other MFS members. However, before employing such models for, e.g., mutational or docking studies, it is essential to develop a measure of their quality. To aid development of such metrics, two disparate MFS members (NupG and GLUT1) have been modeled. In addition, control models were created with shuffled sequences, to mimic poor quality homology models. These models and the template crystal structures have been examined in terms of both static and dynamic indicators of structural quality. Comparison of the behavior of modeled structures with the crystal structures in molecular dynamics simulations provided a metric for model quality. Docking of the inhibitor forskolin to GLUT1 and to a control model revealed significant differences, indicating that we may identify accurate models despite low sequence identity between target sequences and templates.     

New perspectives on the structure and function of transferrins.

Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.     

Deducing hydration sites of a protein from molecular dynamics simulations

Invariant water molecules that are of structural or functional importance to proteins are detected from their presence in the same location in different crystal structures of the same protein or closely related proteins. In this study we have investigated the location of invariant water molecules from MD simulations of ribonuclease A, HIV1-protease and Hen egg white lysozyme. Snapshots of MD trajectories represent the structure of a dynamic protein molecule in a solvated environment as opposed to the static picture provided by crystallography. The MD results are compared to an analysis on crystal structures. A good correlation is observed between the two methods with more than half the hydration sites identified as invariant from crystal structures featuring as invariant in the MD simulations which include most of the functionally or structurally important residues. It is also seen that the propensities of occupying the various hydration sites on a protein for structures obtained from MD and crystallographic studies are different. In general MD simulations can be used to predict invariant hydration sites when there is a paucity of crystallographic data or to complement crystallographic results.     

The structure of apo protein-tyrosine phosphatase 1B C215S mutant: more than just an S --> O change

Protein-tyrosine phosphatases catalyze the hydrolysis of phosphate monoesters via a two-step mechanism involving a covalent phospho-enzyme intermediate. Biochemical and site-directed mutagenesis experiments show that the invariant Cys residue present in the PTPase signature motif (H/V)CX(5)R(S/T) (i.e., C215 in PTP1B) is absolutely required for activity. Mutation of the invariant Cys to Ser results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate-trapping mutants such as the C215S retain, in solution, the structural and binding properties of wild-type PTPases, significant differences have been found in the few studies that have addressed this issue, suggesting that the mutation may lead to structural/conformational alterations in or near the PTP1B binding site. Several crystal structures of apo-WT PTP1B, and of WT- and C215S-mutant PTP1B in complex with different ligands are available, but no structure of the apo-PTP1B C215S has ever been reported. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a surface loop which includes the catalytic Asp) assume a different conformation in the presence or absence of ligand. These observations led to the hypothesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD loop. We report here the structure of the apo-PTP1B C215S mutant, which reveals that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif are in a dramatically different conformation. These results provide a structural basis for the differences in spectroscopic properties and thermodynamic parameters in inhibitor binding observed for the wild-type and mutant enzymes.     

Involvement of putative glutamate receptors in plant defence signaling and NO production

Ionotropic glutamate receptors (iGluRs) are non-selective cation channels permeable to calcium, present in animals and plants. In mammals, glutamate is a well-known neurotransmitter and recently has been recognized as an immunomodulator. As animals and plants share common mechanisms that govern innate immunity with calcium playing a key role in plant defence activation, we have checked the involvement of putative iGluRs in plant defence signaling. Using tobacco cells, we first provide evidence supporting the activity of iGluRs as calcium channels and their involvement in NO production as reported in animals. Thereafter, iGluRs were shown to be activated in response to cryptogein, a well studied elicitor of defence response, and partly responsible for cryptogein-induced NO production. However, other cryptogein-induced calcium-dependent events including anion efflux, H₂O₂ production, MAPK activation and hypersensitive response (HR) did not depend on iGluRs indicating that different calcium channels regulate different processes at the cell level. We have also demonstrated that cryptogein induces efflux of glutamate in the apoplast by exocytosis. Taken together, our results demonstrate for the first time, an involvement of a putative iGluR in plant defence signaling and NO production, by mechanisms that show homology with glutamate mode of action in mammals.     

Intrinsic motions in the N-terminal domain of an ionotropic glutamate receptor detected by fluorescence correlation spectroscopy

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.     

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.