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At least 523 records · Page 29Linked to original sources
PubMed2005

Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors.

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-HT3), gamma-aminobutyric acid (GABA), types A and C, and glycine receptors. Here we present the crystal structure of a remote homolog, AChBP from Bulinus truncatus, which reveals both the conserved structural scaffold and the sites of variation in this receptor family. These include rigid body movements of loops that are close to the transmembrane interface in the receptors and changes in the intermonomer contacts, which alter the pentamer stability drastically. Structural, pharmacological and mutational analysis of both AChBPs shows how 3 amino acid changes in the binding site contribute to a 5-10-fold difference in affinity for nicotinic ligands. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling, and drug design.

Amino Acid Sequence↗
PubMed2005

Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44.

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile(135) of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.

Catalysis↗
PubMed2004

AdoMet radical proteins--from structure to evolution--alignment of divergent protein sequences reveals strong secondary structure element conservation.

Eighteen subclasses of S-adenosyl-l-methionine (AdoMet) radical proteins have been aligned in the first bioinformatics study of the AdoMet radical superfamily to utilize crystallographic information. The recently resolved X-ray structure of biotin synthase (BioB) was used to guide the multiple sequence alignment, and the recently resolved X-ray structure of coproporphyrinogen III oxidase (HemN) was used as the control. Despite the low 9% sequence identity between BioB and HemN, the multiple sequence alignment correctly predicted all but one of the core helices in HemN, and correctly predicted the residues in the enzyme active site. This alignment further suggests that the AdoMet radical proteins may have evolved from half-barrel structures (alphabeta)4 to three-quarter-barrel structures (alphabeta)6 to full-barrel structures (alphabeta)8. It predicts that anaerobic ribonucleotide reductase (RNR) activase, an ancient enzyme that, it has been suggested, serves as a link between the RNA and DNA worlds, will have a half-barrel structure, whereas the three-quarter barrel, exemplified by HemN, will be the most common architecture for AdoMet radical enzymes, and fewer members of the superfamily will join BioB in using a complete (alphabeta)8 TIM-barrel fold to perform radical chemistry. These differences in barrel architecture also explain how AdoMet radical enzymes can act on substrates that range in size from 10 atoms to 608 residue proteins.

Amino Acid Sequence↗
PubMedDate not supplied

The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor.

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.

Animals↗
PubMed2004

Computational analysis of alpha-helical membrane protein structure: implications for the prediction of 3D structural models.

Relatively little has been known about the structure of alpha-helical membrane proteins, since until recently few structures had been crystallized. These limited data have restricted structural analyses to the prediction of secondary structure, rather than tertiary folds. In order to address this, this paper describes an analysis of the 23 available membrane protein structures. A number of findings are made that are of particular relevance to transmembrane helix packing: (1) on average lipid-tail-accessible transmembrane residues are significantly more hydrophobic, less conserved and contain different residue types to buried residues; (2) charged residues are not always buried and, when accessible to membrane lipid tails, few are paired with another charge and instead they often interact with phospholipid head-groups or with other residue types; (3) a significant proportion of lipid-tail-accessible charged and polar residues form hydrogen bonds only with residues one turn away in the same helix (intra-helix); (4) pore-lining residues are usually hydrophobic and it is difficult to distinguish them from buried residues in terms of either residue type or conservation; and (5) information was gained about the proportion of helices that tend to contribute to lining a pore and the resulting pore diameter. These findings are discussed with relevance to the prediction of membrane protein 3D structure.

Databases, Protein↗
PubMed2000

Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data.

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of pl6INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.

Base Sequence↗
PubMed2006

Complete predicted three-dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin superfamily.

Tetraspanins are a superfamily of transmembrane proteins implicated in cellular development, motility, and activation through their interactions with a large range of proteins and with specific membrane microdomains. The complete three-dimensional structure of the tetraspanin CD81 has been predicted by molecular modeling and from the crystallographic structure of the EC2 large extracellular domain. Periodicity of sequence conservation, homology modeling, secondary structure prediction, and protein docking were used. The transmembrane domain appears organized as a four-stranded left-handed coiled coil directly connecting to two helices of the EC2. A smaller extracellular loop EC1 contains a small largely hydrophobic beta-strand that packs in a conserved hydrophobic groove of the EC2. The palmitoylable intracellular N-terminal segment forms an amphipathic membrane-parallel helix. Structural variability occurs mainly in an hypervariable subdomain of the EC2 and in intracellular regions. Therefore, the variable interaction selectivity of tetraspanins originates both from sequence variability within structurally conserved domains and from the occurrence of small structurally variable domains. In CD81 and other tetraspanins, the numerous membrane-exposed aromatic residues are asymmetrically clustered and protrude on one side of the transmembrane domain. This may represent a functional specialization of these two sides for interactions with cholesterol, proteins, or membrane microdomains.

Algorithms↗
PubMed2004

Structure-based functional discovery of proteins: structural proteomics.

The discovery of biochemical and cellular functions of unannotated gene products begins with a database search of proteins with structure/sequence homologues based on known genes. Very recently, a number of frontier groups in structural biology proposed a new paradigm to predict biological functions of an unknown protein on the basis of its three-dimensional structure on a genomic scale. Structural proteomics (genomics), a research area for structure-based functional discovery, aims to complete the protein-folding universe of all gene products in a cell. It would lead us to a complete understanding of a living organism from protein structure. Two major complementary experimental techniques, X-ray crystallography and NMR spectroscopy, combined with recently developed high throughput methods have played a central role in structural proteomics research; however, an integration of these methodologies together with comparative modeling and electron microscopy would speed up the goal for completing a full dictionary of protein folding space in the near future.

Animals↗
PubMed2002

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core.

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.

Animals↗
PubMed2026

Dynamic Protein Structure Paradox: An Integrative Framework for Endpoint-Conditioned Evidentiary Sufficiency in Structure-to-Function Claims.

Accurate coordinates for a represented protein state do not, by themselves, establish activity or any other condition-specific function. This article defines the Dynamic Protein Structure Paradox (DPSP) as the apparent conflict between structural accuracy and functional underdetermination and develops it as an integrative evidentiary assessment framework rather than a new theory or paradigm. The underlying problem has been longstanding, since structural genomics, function annotation, allostery, and disorder research each established that fold does not determine function and that function does not determine fold. DPSP consolidates those results into one endpoint-conditioned rule. Once a measurable endpoint is defined, it assesses four coupled dimensions: relevant-state completeness, context completeness, ensemble or kinetic dependence, and chemical dependence. A rubric rates each dimension as adequate, uncertain, or missing, and a materiality test determines which gaps influence the stated decision. The outcome is one of three mutually exclusive modes of utilization: geometry-led, conditional, or function-measured. The deliverable is a concise evidence statement delineating what the structure supports, which decisive variable remains unmeasured, and what corroboration is necessary. DPSP complements, rather than replaces, existing structural, ensemble, and computational approaches. The framework remains unvalidated, its thresholds are provisional, and the studies necessary to confirm or refute it are specified.

Proteins↗
PubMed1999

Finding local structural similarities among families of unrelated protein structures: a generic non-linear alignment algorithm.

We have developed a generic tool for the automatic identification of regions of local structural similarity in unrelated proteins having different folds, as well as for defining more global similarities that result from homologous protein structures. The computer program GENFIT has evolved from the genetic algorithm-based three-dimensional protein structure comparison program GA_FIT. GENFIT, however, can locate and superimpose regions of local structural homology regardless of their position in a pair of structures, the fold topology, or the chain direction. Furthermore, it is possible to restrict the search to a volume centered about a region of interest (e.g., catalytic site, ligand-binding site) in two protein structures. We present a number of examples to illustrate the function of the program, which is a parallel processing implementation designed for distribution to multiple machines over a local network or to run on a single multiprocessor computer.

Algorithms↗
PubMed1993

Dynamics and structures of DNA: long-range effects of a 16 base-pair (CG)8 sequence on secondary structure.

The effects of inserting 16 base pair (bp) of alternating CG [(CG)8] near the middle of a much longer restriction fragment (1097 bp) are investigated by measuring various properties that are sensitive to secondary and tertiary structure. Results for this fragment are compared with those for a control fragment (1089 bp) with the identical sequence except at the insert. Another fragment (1382 bp), which contains a 296-bp extension at the 5'-end of the 1089-bp control fragment, is also used as a secondary control in some experiments. When the 1097-bp (CG)8 insert fragment is compared with the control fragments in 0.1 M NaCl buffer, the (CG)8 insert is found to induce disproportionately large relative changes in the molar ellipticity at 273 nm ([theta]273), the torsion constant (alpha) measured by fluorescence polarization anisotropy, the optical melting profile, and the susceptibility to S1 nuclease. Estimates of the minimum distance over which the (CG)8 insert alters the secondary structure range from 330 to 550 bp. With increasing NaCl concentration, the 1097-bp insert fragment undergoes a structural transition between 2.0 and 2.5 M that is manifested in the apparent diffusion coefficient (Dplat) from dynamic light scattering at large scattering vector. This transition, which is not exhibited by the control DNAs, is presumed to involve formation of Z-helix at the insert. However, the observed decrease in (Dplat) is attributed to an increase in bending rigidity, which perforce must be globally distributed far beyond the (CG)8 insert per se. In 4.25 M NaCl (but not in 0.1 M NaCl), the addition of 1 ethidium dye per 300 bp induces an extensive structural transition in the 1097 bp (CG)8 insert fragment. This transition, which also is not exhibited by the control DNAs, significantly decreases the bending rigidity, doubles [theta]273, and takes place on a time scale of a few days. Removal of ethidium and salt by dialysis vs 0.1 M NaCl buffer restores the original properties of the 1097-bp (CG)8 insert fragment. The present results are consistent with a (fluctuating, long-range) description of the secondary structure in which a given short sequence transiently fluctuates among two or more distinct secondary structures that extend over much larger domains of variable position and size, and whose relative stabilities depend on distant as well as close-lying base pairs.

Base Composition↗
PubMed1993

Prediction of the three-dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H-protein from the pea leaf glycine cleavage system: a new automated method for the prediction of protein tertiary structure.

A new, automated, knowledge-based method for the construction of three-dimensional models of proteins is described. Geometric restraints on target structures are calculated from a consideration of homologous template structures and the wider knowledge base of unrelated protein structures. Three-dimensional structures are calculated from initial partly folded states by high-temperature molecular dynamics simulations followed slow cooling of the system (simulated annealing) using nonphysical potentials. Three-dimensional models for the biotinylated domain from the pyruvate carboxylase of yeast and the lipoylated H-protein from the glycine cleavage system of pea leaf were constructed, based on the known structures of two lipoylated domains of 2-oxo acid dehydrogenase multienzyme complexes. Despite their weak sequence similarity, the three proteins are predicted to have similar three-dimensional structures, representative of a new protein module. Implications for the mechanisms of posttranslational modification of these proteins and their catalytic function are discussed.

Amino Acid Oxidoreductases↗
PubMed1998

The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

Animals↗
PubMed2005

Dependency between consecutive local conformations helps assemble protein structures from secondary structures using Go potential and greedy algorithm.

Discretization of protein conformational space and fragment assembly methods simplify the search of native structures. These methods, mostly of Monte Carlo and genetic-type, do not exploit, however, the fact that short fragments describing consecutive parts of proteins are conformation-dependent. Yet, this information should be useful in improving ab initio and comparative protein structure modeling. In a preliminary study, we have assessed the possibility of using greedy algorithms for protein structure reconstruction based on the assembly of fragments of four-residue length. Greedy algorithms differ from Monte Carlo and genetic approaches in that they grow a polypeptide chain one fragment after another. Here, we move one step further in complexity, and provide strong evidence that the dependence between consecutive local conformations during assembly makes possible the reconstruction of protein structures from their secondary structures using a Go potential. Overall our procedure can reproduce 20 protein structures of 50-164 amino acids within 2.7 to 6.5 A RMSd and is able to identify native topologies for all proteins, although some targets are stabilized by very long-range interactions.

Algorithms↗
PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. II. Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex.

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations. The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical. The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell. Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses. Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure. The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Bacteriophage phi 6↗
PubMed1995

Structure determination of the biliverdin apomyoglobin complex: crystal structure analysis of two crystal forms at 1.4 and 1.5 A resolution.

Crystal structure determinations of two orthorhombic (P2(1)2(1)2(1)) crystal modifications of the biliverdin apomyoglobin complex are described. The two structures were determined by X-ray diffraction at 100 K to a resolution of 1.5 A and 1.4 A. Both crystal forms were grown by hanging-drop techniques, using phosphate as precipitant. The structures were solved by molecular replacement and refined to final R-values of 19.4% and 21.2%. Both structures are very similar with respect to the binding site and the conformation of the biliverdin chromophore, which occurs in a (P) helical conformation. It is located within the heme pocket, very close in position and orientation to the heme binding site in myoglobin. Two water molecules not present in the crystal structure of myoglobin are sequestered within the heme pocket in the biliverdin-apomyoglobin complex, and they are engaged in hydrogen bonding to the biliverdin and to the protein. Comparison with structural results from an earlier NMR study of the same complex shows good agreement.

Apoproteins↗
PubMed1999

Prediction of protein tertiary structure to low resolution: performance for a large and structurally diverse test set.

We report the tertiary structure predictions for 95 proteins ranging in size from 17 to 160 residues starting from known secondary structure. Predictions are obtained from global minimization of an empirical potential function followed by the application of a refined atomic overlap potential. The minimization strategy employed represents a variant of the Monte Carlo plus minimization scheme of Li and Scheraga applied to a reduced model of the protein chain. For all of the cases except beta-proteins larger than 75 residues, a native-like structure, usually 4-6 A root-mean-square deviation from the native, is located. For beta-proteins larger than 75 residues, the energy gap between native-like structures and the lowest energy structures produced in the simulation is large, so that low RMSD structures are not generated starting from an unfolded state. This is attributed to the lack of an explicit hydrogen bond term in the potential function, which we hypothesize is necessary to stabilize large assemblies of beta-strands.

Algorithms↗