Three-dimensional models of glutamate receptors.
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Amino Acid Sequence↗
Biomedical subjects
M J Sutcliffe
Publications and source records attributed to M J Sutcliffe.
Probing the stabilizing role of C-terminal residues in trimethylamine dehydrogenase.
In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotein, the C-terminal 17 residues of each subunit (residues 713-729) embrace residues on the other subunit. The role of this unusual mode of interaction at the subunit interface was probed by isolating three mutant forms of trimethylamine dehydrogenase in which the C-terminus of the enzyme was deleted by five residues [delta(725-729], 10 residues [delta(720-729)] and 17 residues [delta(713-729)]. The solution properties and conformational states of the three mutant enzymes were investigated using optical, fluorescence and circular dichroism spectroscopies, ANS binding and a novel and conformationally sensitive hydrodynamic method. The data reveal that sequential deletion of the C-terminus of trimethylamine dehydrogenase does not affect significantly dimer stability or the overall structural integrity of the enzyme. However, deletion of the C-terminus severely compromises, but does not abolish, the ability of the enzyme to become covalently coupled with the redox cofactor FMN in the active site, located over 20 A from the C-terminus. Hydrodynamic studies reveal minor conformational changes in the deletion mutants that lead to a more compact enzyme structure. These conformational changes are probably transmitted to the active site via altering the interaction of the C-terminus with the second helix in the beta/alpha barrel of trimethylamine dehydrogenase, leading to poor flavinylation during the folding of the enzyme and assembly with FMN.
Bacteria↗
Assignment1 of microsomal epoxide hydrolase (EPHX1) to human chromosome 1q42.1 by in situ hybridization.
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Chromosome Mapping↗
Three-dimensional models of glutamate receptors.
Structural models of glutamate receptors have been produced as part of a multidisciplinary study of neuronal function--both ligand/receptor interactions and ion transport--at the atomic level. The models have concentrated on the agonist binding and transmembrane domains of ionotropic (i.e. ligand-gated) glutamate receptors (iGluRs), and have aided our understanding of the molecular determinants of (1) ligand binding and (2) channel activity. The model building process involved a combination of homology modelling, distance geometry, molecular mechanics, protein-ligand and protein-protein docking, electrostatic calculations and manual adjustment, in conjunction with restraints from site-directed mutagenesis, ligand binding and electrophysiological studies. The initial models were used to produce hypotheses which were tested experimentally; these models have been subsequently refined as part of an extremely effective multidisciplinary study using an iterative molecular modelling/experimental verification cycle in which restraints derived from experimental studies are used at all stages, and the findings from one round of modelling are used as restraints in the next. By studying a variety of agonists and antagonists, details have been built up of (1) those residues involved in ligand binding and (2) the role of agonist binding (i.e. agonist-induced conformational change) in channel gating. The models also aid our understanding of the conductance properties of the channels.
Amino Acid Sequence↗
Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase.
The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.
Ascorbate Peroxidases↗
Definition of the switch surface in the solution structure of Cdc42Hs.
Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins. Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement. These structures show overall similarity to those of other GTP-binding proteins, with some exceptions. The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an alpha-helix in switch II. The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the beta4-alpha3 loop. The insert forms one edge of a "switch surface" and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP. These studies indicate the insert region forms a stable invariant "footrest" for docking of regulatory and effector proteins.
Amino Acid Sequence↗
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation.
Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.
Bacillus megaterium↗
An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: kinetics of transfer in wild-type and mutant complexes.
In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.
Bacteria↗
OLDERADO: on-line database of ensemble representatives and domains. On Line Database of Ensemble Representatives And DOmains.
In cases where the structure of a single protein is represented by an ensemble of conformations, there is often a need to determine the common features and to choose a "representative" conformation. This occurs, for example, with structures determined by NMR spectroscopy, analysis of the trajectory from a molecular dynamics simulation, or an ensemble of structures produced by comparative modeling. We reported previously automatic methods for (1) defining the atoms with low spatial variance across an ensemble (i.e., the "core" atoms) and the domains in which these atoms lie, and (2) clustering an ensemble into conformationally related subfamilies. To extend the utility of these methods, we have developed a freely available server on the World Wide Web at http:/(/)neon.chem.le.ac.uk/olderado/. This (1) contains an automatically generated database of representative structures, core atoms, and domains determined for 449 ensembles of NMR-derived protein structures in the Protein Data Bank (PDB) in May 1997, and (2) allows the user to upload a PDB-formatted file containing the coordinates of an ensemble of structures. The server returns in real time: (1) information on the residues constituting domains: (2) the structures that constitute each conformational subfamily; and (3) an interactive java-based three-dimensional viewer to visualise the domains and clusters. Such information is useful, for example, when selecting conformations to be used in comparative modeling and when choosing parts of structures to be used in molecular replacement. Here we describe the OLDERADO server.
Computer Communication Networks↗
An automated approach for defining core atoms and domains in an ensemble of NMR-derived protein structures.
A single NMR-derived protein structure is usually deposited as an ensemble containing many structures, each consistent with the restraint set used. The number of NMR-derived structures deposited in the Protein Data Bank (PDB) is increasing rapidly. In addition, many of the structures deposited in an ensemble exhibit variation in only some regions of the structure, often with the majority of the structure remaining largely invariant across the family of structures. Therefore it is useful to determine the set of atoms whose positions are 'well defined' across an ensemble (also known as the 'core' atoms). We have developed a computer program, NMRCORE, which automatically defines (i) the core atoms, and (ii) the rigid body(ies), or domain(s), in which they occur. The program uses a sorted list of the variances in individual dihedral angles across the ensemble to define the core, followed by the automatic clustering of the variances in pairwise inter-atom distances across the ensemble to define the rigid body(ies) which comprise the core. The program is freely available via the World Wide Web (http://neon.chem.le.ac.uk/nmrcore/).
Algorithms↗
Ligand binding to integrin alphaIIbbeta3 is dependent on a MIDAS-like domain in the beta3 subunit.
Substitution of beta3 residue Asp119, Ser121, or Ser123 results in a loss of the ligand binding function of integrin alphaIIbbeta3. Homologous residues in other integrin beta subunits are similarly critical for ligand binding function. This DXSXS motif is also present in the I domain of certain integrin alpha subunits, where it constitutes a portion of the unique metal ion-dependent adhesion site (MIDAS). In this report, we have utilized the crystal structure of the recombinant alphaM I domain to produce a three-dimensional model of the homologous region in the integrin beta3 subunit. We performed mutagenesis of candidate amino acid residues predicted from this model to be involved in cation coordination and ligand binding. We report the identification of Asp217 and Glu220 as residues essential for the ligand binding function of alphaIIbbeta3. Alanine substitution of these residues did not affect receptor expression but abolished the binding of activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies. In our proposed model, beta3 Asp217 is analogous to a metal-coordinating residue in the alphaM MIDAS domain, while Glu220 does not correspond to a functional MIDAS domain residue. Substitution of the highly conserved beta3 residue Thr197 corresponding to a critical MIDAS metal-coordinating Thr residue did not affect ligand binding function, suggesting that this region of beta3 adopts a structure that is very similar to but not identical to that of the MIDAS domain. These data support a functional linkage between these two sequences and further define a common feature of ligand binding to integrins.
Amino Acid Sequence↗
Crystal structure of a PDZ domain.
PDZ domains (also known as DHR domains or GLGF repeats) are approximately 90-residue repeats found in a number of proteins implicated in ion-channel and receptor clustering, and the linking of receptors to effector enzymes. PDZ domains are protein-recognition modules; some recognize proteins containing the consensus carboxy-terminal tripeptide motif S/TXV with high specificity. Other PDZ domains form homotypic dimers: the PDZ domain of the neuronal enzyme nitric oxide synthase binds to the PDZ domain of PSD-95, an interaction that has been implicated in its synaptic association. Here we report the crystal structure of the third PDZ domain of the human homologue of the Drosophila discs-large tumour-suppressor gene product, DlgA. It consists of a five-stranded antiparallel beta-barrel flanked by three alpha-helices. A groove runs over the surface of the domain, ending in a conserved hydrophobic pocket and a buried arginine; we suggest that this is the binding site for the C-terminal peptide.
Adaptor Proteins, Signal Transducing↗
A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding.
The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.
Amino Acid Sequence↗
Monosomy 6q1: syndrome delineation.
We report on a girl with a de novo 6q1 interstitial deletion. To our knowledge, this is the second reported case with a deletion of 6q11-q15. We review the phenotype of monosomy 6q1. Our patient has manifestations similar to others with monosomy 6q1 including mental deficiency, growth retardation, short neck, and minor facial anomalies.
Abnormalities, Multiple↗
NMR studies of the mode of binding of corepressors and inducers to Escherichia coli trp repressor.
The binding of the corepressors tryptophan and 5-methyltryptophan and of the inducers 3-indolepropionate, 3-indoleacrylate and 5-methylindole to the Escherichia coli trp repressor have been studied by 1H-NMR spectroscopy. Identification of the resonances of the protons of bound ligands and their NOEs to protons of the protein (measured as transferred NOE) was greatly facilitated by the use of samples of the protein in which the hydrogens of all residues except alanine, isoleucine and threonine was replaced by deuterium. Chemical-shift changes of protein-backbone resonances and side-chain-amide resonances on ligand binding were measured with generally or selectively 15N-labelled protein. The patterns of changes in the chemical shifts of protein resonances and, particularly, ligand resonances distinguish the corepressors from the inducers, indicating, in agreement with earlier work, that corepressors and inducers bind to the protein in different ways. The NOEs observed for the bond ligands have been used to determine the position of the ligands in the crystallographically determined binding site, by means of a simulated-annealing molecular-dynamics protocol. The structures obtained show that the orientation in the binding site of the indole rings of tryptophan and 5-methyltryptophan and of 3-indolepropionate and 3-indoleacrylate differ by approximately 180 degrees in solution (in agreement with the crystallographic data for complexes of the trp repressor with tryptophan or with 3-indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE are discussed.
Bacterial Proteins↗
Fanconi anemia in brothers initially diagnosed with VACTERL association with hydrocephalus, and subsequently with Baller-Gerold syndrome.
Two brothers with presumed Baller-Gerold syndrome, one of whom was previously diagnosed with the association of vertebral, cardiac, renal, limb anomalies, anal atresia, tracheo-esophageal fistula (VACTERL) association with hydrocephalus, were evaluated for chromosome breakage because of severe thrombo cytopenia in one of them. Spontaneous and clastogen-induced breakage was markedly increased in both patients as compared to control individuals. Clinical manifestations and chromosome breakage, consistent with Fanconi anemia, in patients with a prior diagnosis of either Baller-Gerold syndrome, reported earlier in one other patient [Farrell et al., 1994: Am J Med Genet 50:98-99], or with VACTERL association with hydrocephalus, recently reported in 3 patients [Toriello et al., 1991: Proc Greenwood Genet Center 11:142; Porteus et al., 1992: Am J Med Genet 43:1032-1034], underline the clinical heterogeneity of Fanconi anemia and raise the question of whether these syndromes are distinct disorders or phenotypic variations of the same disease.
Abnormalities, Multiple↗
Three-dimensional models of non-NMDA glutamate receptors.
Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate receptor, GluR6; and a low-molecular-weight kainate receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains of the proteins that bind the neurotransmitter glutamate and that form the ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive modeling, and known constraints. The models indicate new potential interactions in the extracellular domain between protein and agonists, and suggest that the transition from the "closed" to the "open" state involves the movement of a conserved positive residue away from, and two conserved negative residues into, the extracellular entrance to the pore upon binding. As a first approximation, the ion channel domain was modeled with a structure comprising a central antiparallel beta-barrel that partially crosses the membrane, and against which alpha-helices from each subunit are packed; a third alpha-helix packs against these two helices in each subunit. Much, but not all, of the available data were consistent with this structure. Modifying the beta-barrel to a loop-like topology produced a model consistent with available data.
Amino Acid Sequence↗