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PubMed2002

X-ray crystal structures of reduced rubrerythrin and its azide adduct: a structure-based mechanism for a non-heme diiron peroxidase.

Rubrerythrin (Rbr) is a 44-kDa homodimeric protein, found in many air-sensitive bacteria and archaea, which contains a unique combination of a rubredoxin-like [Fe(SCys)(4)] site and a non-sulfur, oxo/dicarboxylato-bridged diiron site. The diiron site structure resembles those found in O2-activating diiron enzymes. However, Rbr instead appears to function as a hydrogen peroxide reductase (peroxidase). The diferrous site in all-ferrous Rbr (Rbr(red)) shows a much greater reactivity with H2O2 than does the diferric site in all-ferric Rbr (Rbr(ox)), but only the latter structure has been reported. Here we report the X-ray crystal structures of the recombinant Rbr(red) from the sulfate reducing bacterium, Desulfovibrio vulgaris, as well as its azide adduct (Rbr(red)N3). We have also redetermined the structure of Rbr(ox) to a higher resolution than previously reported. The structural differences between Rbr(ox) and Rbr(red) are localized entirely at the diiron site. The most striking structural change upon reduction of the diferric to the diferrous site of Rbr is a 1.8-A movement of one iron away from a unique glutamate carboxylate ligand and toward a trans-disposed histidine side chain, which replaces the glutamate as a ligand. This movement increases the inter-iron distance from 3.3 to 4 A. Rbr(red)N(3) shows this same iron movement and His-->Glu ligand replacement relative to Rbr(ox), and, in addition, an azide coordinated to the diiron site in a cis mu-1,3 fashion, replacing two solvent ligands in Rbr(red). Relative to those in O2-activating enzymes, the bridging carboxylate ligation of the Rbr diiron site is less flexible upon diferric/diferrous interconversion. The diferrous site is also much more rigid, symmetrical, and solvent-exposed than those in O2-activating enzymes. On the basis of these unique structural features, a mechanism is proposed for facile reduction of hydrogen peroxide by Rbr involving a cis mu-eta(2) H2O2 diferrous intermediate.

Azides↗
PubMed2005

Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH.

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.

Chromatography, Gel↗
PubMed2003

Molecular modeling of the three-dimensional structure of dopamine 3 (D3) subtype receptor: discovery of novel and potent D3 ligands through a hybrid pharmacophore- and structure-based database searching approach.

The dopamine 3 (D3) subtype receptor has been implicated in several neurological conditions, and potent and selective D3 ligands may have therapeutic potential for the treatment of drug addiction, Parkinson's disease, and schizophrenia. In this paper, we report computational homology modeling of the D3 receptor based upon the high-resolution X-ray structure of rhodopsin, extensive structural refinement in the presence of explicit lipid bilayer and water environment, and validation of the refined D3 structural models using experimental data. We further describe the development, validation, and application of a hybrid computational screening approach for the discovery of several classes of novel and potent D3 ligands. This computational approach employs stepwise pharmacophore and structure-based searching of a large three-dimensional chemical database for the identification of potential D3 ligands. The obtained hits are then subjected to structural novelty screening, and the most promising compounds are tested in a D3 binding assay. Using this approach we identified four compounds with K(i) values better than 100 nM and eight compounds with K(i) values better than 1 microM out of 20 compounds selected for testing in the D3 receptor binding assay. Our results suggest that the D3 structural models obtained from this study may be useful for the discovery and design of novel and potent D3 ligands. Furthermore, the employed hybrid approach may be more effective for lead discovery from a large chemical database than either pharmacophore-based or structure-based database screening alone.

Algorithms↗
PubMed2004

Prediction of 3-D structures of fucose-binding proteins and structural analysis of their interaction with ligands.

The importance of fucose-binding proteins stems from the presence of fucose as terminal sugars in H and Lewis (a) blood groups. Recently, the structure of a complex between Anguilla anguilla agglutinin (AAA) and alpha-L-fucose has been worked out at 1.9 A resolution. The structure of AAA characterizes the novel fold of an entire lectin family. In the present study, molecular modeling techniques have been used to identify new proteins that can provide a similar fucose binding module in the newly discovered genomic sequences using the above mentioned structural information. We modeled 3-D structures of three such proteins, namely, ebiP5322 protein of Anopheles gambiae, a pentraxin of Xenopus laevis, and the fw gene product of Drosophila melanogaster. alpha-L-fucose was docked in the binding pockets of the modeled structures followed by energy minimization and molecular dynamic runs to obtain the most probable structures of the complexes. Properties of these modeled complexes were studied to examine the nature of physicochemical forces involved in the complex formation and compared with AAA-alpha-L-fucose complex. It was found that ebiP5322 protein of A. gambiae and the pentraxin of X. laevis can provide a fucose-binding fold similar to AAA. We studied structures of four protein-fucose complexes to examine the electrostatic potential surfaces around the binding site and concluded that a highly positive-charged surface was not a necessary condition of fucose-binding.

Animals↗
PubMed1998

The solution NMR structure of a blue-green algae hepatotoxin, microcystin-RR--a comparison with the structure of microcystin-LR.

The microcystin-RR structures are compared with the structures of microcystin-LR in solution as well as in the crystal structure of the complex with protein phosphatase. The gross structures of the two peptides are similar, but with a more accentuated and compact saddle structure for microcystin-RR. The structural differences affect the hydrogen-bond pattern in the peptides and the location of the side chain of N-methyldehydroalanine, both of which are important for the ability of the peptide to form a tight complex with protein phosphatase. These structural differences may contribute to the observed differences in toxicity of microcystin-RR and microcystin-LR.

Cyanobacteria↗
PubMed1995

Crystal structures of recombinant rat cathepsin B and a cathepsin B-inhibitor complex. Implications for structure-based inhibitor design.

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.

Amino Acid Chloromethyl Ketones↗
PubMed2002

The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR.

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Amino Acid Sequence↗
PubMed2000

Crystal structure of recombinant native SDF-1alpha with additional mutagenesis studies: an attempt at a more comprehensive interpretation of accumulated structure-activity relationship data.

Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.

Amino Acid Motifs↗
PubMed2006

Protein structural motif prediction in multidimensional phi-psi space leads to improved secondary structure prediction.

A significant step towards establishing the structure and function of a protein is the prediction of the local conformation of the polypeptide chain. In this article, we present systems for the prediction of three new alphabets of local structural motifs. The motifs are built by applying multidimensional scaling (MDS) and clustering to pair-wise angular distances for multiple phi-psi angle values collected from high-resolution protein structures. The predictive systems, based on ensembles of bidirectional recurrent neural network architectures, and trained on a large non-redundant set of protein structures, achieve 72%, 66%, and 60% correct motif prediction on an independent test set for di-peptides (six classes), tri-peptides (eight classes) and tetra-peptides (14 classes), respectively, 28-30% above baseline statistical predictors. We then build a further system, based on ensembles of two-layered bidirectional recurrent neural networks, to map structural motif predictions into a traditional 3-class (helix, strand, coil) secondary structure. This system achieves 79.5% correct prediction using the "hard" CASP 3-class assignment, and 81.4% with a more lenient assignment, outperforming a sophisticated state-of-the-art predictor (Porter) trained in the same experimental conditions. The structural motif predictor is publicly available at: http://distill.ucd.ie/porter+/.

Amino Acid Motifs↗
PubMed2003

Structure-activity relationship of oxadiazoles and allylic structures in the Ames test: an industry screening approach.

In recent years genotoxicity testing has become more and more important in the process of early screening for potential development compounds. In the case that a pharmacologically interesting structure is found to be positive in an in vitro mutagenicity test a straightforward approach starts by sorting out what substructure is responsible for the activity observed in the test. The Ames test is a rapid, convenient test system which has been effectively used in structure-activity relationship studies for mutagenicity, since it can rapidly establish differences in the mutagenic action of isomers and chemical analogs. The lead compound with a benzodiazepine-like structure and close analogs exhibited weak, but unequivocal positive effects in the Ames test (strains TA1535 and TA 100) after metabolic activation by rat liver homogenate fraction (S9). To identify substances within this class of compounds devoid of mutagenic liability an extensive structure-activity investigation was undertaken. More than 50 compounds were tested in the two critical bacterial strains, using a standard plate incorporation and a preincubation modification. It quickly became evident that the benzodiazepine structure was not involved. First hints that the allyl side chain were responsible for the Ames activity had to be refined in a more complex, but clear-cut structure-activity relationship during the course of the experiments. It was shown that all compounds with an allyl side chain, independent of the heterocycle, but surprisingly also all compounds with a specific arrangement of the heteroatoms in the oxadiazole ring, showed positive effects in at least one strain. Based on these investigations it was possible to select pharmacologically active structures without mutagenic liability.

Mutagenicity Tests↗
PubMed2006

ProKware: integrated software for presenting protein structural properties in protein tertiary structures.

Protein tertiary structure plays an essential role in deciphering protein functions, especially protein structural properties, including domains, active sites and post-translational modifications. These properties typically yield useful clues for understanding protein functions. This work presents an integrated software, named ProKware, that presents protein structural properties in protein tertiary structures, such as domains, functional sites, families, active sites, binding sites, post-translational modifications and domain-domain interaction. Using this web-based and Windows-based interface, users can manipulate and visualize three-dimensional protein structures, as well as the supported structural properties that are curated in the protein knowledge database. ProKware is an effective and convenient solution for investigating protein functions and structural relationships. This software can be accessed on the internet at http://ProKware.mbc.nctu.edu.tw/.

Binding Sites↗
PubMed1998

Quantitative structure-function and structure-stability relationships of purposely modified proteins.

Quantitative structure-function relationships (QSFR) and quantitative structure-stability relationships (QSSR) analyses are described here. The objective of these analyses is to investigate and quantitatively describe the effect of the changes in structure of protein on its function or stability. During the analysis, the structural and physico-chemical properties of the amino acid residues are related to activity or stability data derived for the group of proteins containing systematic substitutions at certain positions. Four examples of the application of these analyses on the data obtained with proteins modified by site-directed mutagenesis experiments are provided. Structure-function relationships were studied for 15 mutants in position 172 of the haloalkane dehalogenase and 19 mutants in position 222 of the subtilisin, while the structure-stability relationships were investigated for 13 mutants in position 157 of phage T4 lysozyme and 18 mutants in position 49 of alpha-subunits tryptophan synthase. A total of 402 molecular descriptors derived from AAindex database were used to quantify amino acid properties and the multivariate statistical technique--partial least squares projections to latent structures--was used to identify those of them which are important for explanation of the activity and stability data. Quantitative models were developed and internally validated for every data set. The possibilities for further development of both analyses and their application for predictive and analytical purposes in protein engineering research are discussed.

Glutamic Acid↗
PubMed2002

The 1.35 A structure of cadmium-substituted TM-3, a snake-venom metalloproteinase from Taiwan habu: elucidation of a TNFalpha-converting enzyme-like active-site structure with a distorted octahedral geometry of cadmium.

The crystal structure of TM-3, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.35 A resolution with resultant R and R(free) values of 0.181 and 0.204, respectively. The overall structure of TM-3 is an oblate ellipsoid that contains three disulfide crosslinks, Cys118-Cys197, Cys159-Cys181 and Cys161-Cys164. It exhibits the typical structural features of SVMPs and is closely related to the structure of the catalytic proteinase domain of TNFalpha-converting enzyme (TACE). In the present structure, the essential catalytic zinc ion was found to be replaced by a cadmium ion during crystallization, as revealed by atomic absorption analysis and X-ray data. This cadmium ion is bound to six ligands, including three conserved histidines and three water molecules, displaying the coordination geometry of a distorted octahedron. One of the water molecules is proposed to play the role of stabilizing the tetrahedral intermediate during the catalysis of SVMPs. The putative S'(1) specificity pocket of TM-3 is relatively shallow, in contrast to the deep pockets of adamalysin II, atrolysin C and H(2)-proteinase, but is similar to those in acutolysin A and TACE. The shallow pocket is a consequence of the presence of the non-conserved disulfide bond Cys159-Cys181 and the residue Gln174 at the bottom of the S'(1) pocket. The results indicate that the active-site structure of TM-3, among the know structures of SVMPs examined thus far, is most similar to that of TACE owing to their close disulfide configurations and the S'(1) specificity pocket.

ADAM Proteins↗
PubMed1999

Structure of an insulin dimer in an orthorhombic crystal: the structure analysis of a human insulin mutant (B9 Ser-->Glu).

The structure of human insulin mutant B9 (Ser-->Glu) was determined by an X-ray crystallographic method at 2.5 A resolution with an R factor of 0.165 under non-crystallographic restraints. The crystals were grown at low pH (<3.8) and belong to the orthorhombic P2(1)2(1)2(1) space group with unit-cell dimensions a = 44.54, b = 46.40, c = 51.85 A and one dimer per asymmetric unit without further aggregation. The structure in this crystal form can be regarded as a model for a discrete insulin dimer and displays the following features compared with the structure of 2Zn insulin. (i) The overall dimer is expanded and more symmetric. The two A chains are about 2 A more distant from each other, while the two B chains are about 0.8 A further apart. Both monomers are more similar to molecule 1 than molecule 2 of the 2Zn insulin dimer. (ii) The dimer structure is stabilized by protonation and neutralization of the carboxyl groups at lower pH and, in addition, by formation of a hydrogen-bond network among the side chains of residues GluB9, HisB13 and HisB10 on the dimer-forming surface of both monomers, resulting from a structural rearrangement. (iii) The B-chain amino-terminal segment is in an open state (O state), i.e. a state different from the well known R and T states found in the insulin hexamer. In the O state, the B-chain N-terminal segment is in an extended conformation and is detached from the rest of the molecule. This conformational state has also been observed in the monomeric crystal structure of despentapeptide (B26-B30) and desheptapeptide (B24-B30) insulin, as well as in the solution structure of an engineered insulin monomer. It suggests that the O state may be the characteristic conformation of insulin in lower aggregation forms and may be relevant to the formation of insulin fibrils. In addition, based on the crystallization process, the smallest possible building blocks of insulin crystal are also discussed.

Amino Acid Substitution↗
PubMed2003

Structural genomics: computational methods for structure analysis.

The success of structural genomics initiatives requires the development and application of tools for structure analysis, prediction, and annotation. In this paper we review recent developments in these areas; specifically structure alignment, the detection of remote homologs and analogs, homology modeling and the use of structures to predict function. We also discuss various rationales for structural genomics initiatives. These include the structure-based clustering of sequence space and genome-wide function assignment. It is also argued that structural genomics can be integrated into more traditional biological research if specific biological questions are included in target selection strategies.

Amino Acid Motifs↗
PubMed2000

Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)--identification of structural features important for the lethal action of snake venom cardiotoxins.

The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel beta-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct "finger-shaped" projection comprising of nonpolar residues. The occurrence of the nonpolar "finger-shaped" projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the "finger-shaped" projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar "finger-shaped" projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.

Amino Acid Sequence↗
PubMed2004

Structural characterization of the human Nogo-A functional domains. Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration.

The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury. Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration. So far, no structural information is available for Nogo-A or any of its structural domains. We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli. CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble. Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration. Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment. The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol. The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands. Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration.

Amino Acid Sequence↗
PubMed2006

Prediction of protein continuum secondary structure with probabilistic models based on NMR solved structures.

BACKGROUND: The structure of proteins may change as a result of the inherent flexibility of some protein regions. We develop and explore probabilistic machine learning methods for predicting a continuum secondary structure, i.e. assigning probabilities to the conformational states of a residue. We train our methods using data derived from high-quality NMR models. RESULTS: Several probabilistic models not only successfully estimate the continuum secondary structure, but also provide a categorical output on par with models directly trained on categorical data. Importantly, models trained on the continuum secondary structure are also better than their categorical counterparts at identifying the conformational state for structurally ambivalent residues. CONCLUSION: Cascaded probabilistic neural networks trained on the continuum secondary structure exhibit better accuracy in structurally ambivalent regions of proteins, while sustaining an overall classification accuracy on par with standard, categorical prediction methods.

Algorithms↗