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PubMedDate not supplied

The alpha-helix as seen from the protein tertiary structure: a 3-D structural classification.

Helices are selected from globular protein structures defined at high resolution by X-ray analysis. We cluster alpha-helices in two ways: according to their position in the tertiary structure by considering patterns of solvent inaccessible residues and according to the arc of the solvent inaccessible face. For each class of helices we have defined propensities for amino acids at each position; these can be used to calculate templates for recognition of a member of that class. The analysis provides a basis for the prediction of alpha-helices and estimation of their approximate position in a protein tertiary structure. It also provides an approach to estimating the probability of finding amino acid sequences as helices in solution and in a folded protein, thus indicating those helices that might be involved in nucleation of protein folding.

Amino Acid Sequence↗
PubMed2004

Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

Bacillus↗
PubMed2005

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site.

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.

Amino Acid Motifs↗
PubMed2005

Crystal structures of mouse autocrine motility factor in complex with carbohydrate phosphate inhibitors provide insight into structure-activity relationship of the inhibitors.

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is identical to the extracellular cytokines neuroleukin and maturation factor and, interestingly, to the intracellular enzyme phosphoglucose isomerase. The cytokine activity of AMF is inhibited by carbohydrate phosphate compounds as they compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven transmembrane helix protein. Here, we report the first comprehensive high-resolution crystal structure analyses of the inhibitor-free form and the eight types of inhibitor (phosphate, erythrose 4-phosphate (E4P), arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P), 6-phosphogluconic acid (6PGA), fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), or mannose 6-phosphate (M6P)) complexes of mouse AMF (mAMF). We assayed the inhibitory activities of these inhibitors against the cytokine activity of mAMF. The inhibitory activities of the six-carbon sugars (G6P, F6P, M6P, and 6PGA) were found to be significantly higher than those of the four or five-carbon sugars (E4P or A5P). The inhibitory activities clearly depend on the length of the inhibitor molecules. A structural comparison revealed that a water-mediated hydrogen bond between one end of the inhibitor and a rigid portion of the protein surface in the shorter-chain inhibitor (E4P) complex is replaced by a direct hydrogen bond in the longer-chain inhibitor (6PGA) complex. Thus, to obtain a new compound with higher inhibitory activities against AMF, water molecules at the inhibitor binding site of AMF should be replaced by a functional group of inhibitors in order to introduce direct interactions with the protein surface. The present structure-activity relationship studies will be valuable not only for designing more effective AMF inhibitors but also for studying general protein-inhibitor interactions.

Animals↗
PubMed2006

Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms.

14-3-3 proteins are a ubiquitous class of regulatory proteins found in all eukaryotic cells and were the first class of molecules to be recognized as discrete phosphoserine/threonine binding modules. 14-3-3 proteins bind a large number of different substrates to regulate a wide array of cellular signaling events including cell cycle progression and DNA damage responses, programmed cell death, cytoskeletal dynamics, transcriptional control of gene expression, as well as processes directly related to cancer progression. In this review, the structural basis of phosphorylation-dependent binding of 14-3-3 to peptide and protein ligands is discussed along with mechanisms that govern how 14-3-3 regulates the function of its bound ligands. The X-ray crystal structures of all human 14-3-3 proteins bound to peptides have now been solved. Here, we use structural comparisons between isoforms as a framework for discussion of ligand binding by 14-3-3 as well as the mechanisms through which post-translational modification of the different isoforms alters their function.

14-3-3 Proteins↗
PubMed2005

Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics.

A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.

Binding Sites↗
PubMed2002

Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 A resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

Bacterial Proteins↗
PubMed2003

Solution structure of a circular-permuted variant of the potent HIV-inactivating protein cyanovirin-N: structural basis for protein stability and oligosaccharide interaction.

The high-resolution solution structure of a monomeric circular permuted (cp) variant of the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by NMR. Comparison with the wild-type (wt) structure revealed that the observed loss in stability of cpCV-N compared to the wt protein is due to less favorable packing of several residues at the pseudo twofold axis that are responsible for holding the two halves of the molecule together. In particular, the N and C-terminal amino acid residues exhibit conformational flexibility, resulting in fewer and less favorable contacts between them. The important hydrophobic and hydrogen-bonding network between residues W49, D89, H90, Y100 and E101 that was observed in wt CV-N is no longer present. For instance, Y100 and E101 are flexible and the tryptophan side-chain is in a different conformation compared to the wt protein. The stability loss amounts to approximately 2kcal/mol and the mobility of the protein is evident by fast amide proton exchange throughout the chain. Mutation of the single proline residue to glycine (P52G) did not substantially affect the stability of the protein, in contrast to the finding for wtCV-N. The binding of high-mannose type oligosaccharides to cpCV-N was also investigated. Similar to wtCV-N, two carbohydrate-binding sites were identified on the protein and the Man alpha1-->2Man linked moieties on the sugar were delineated as binding epitopes. Unlike in wtCV-N, the binding sites on cpCV-N are structurally similar and exhibit comparable binding affinities for the respective sugars. On the basis of the studies presented here and previous results on high-mannose binding to wtCV-N, we discuss a model for the interaction between gp120 and CV-N.

Amino Acid Sequence↗
PubMed2003

Crystal structure of thermostable aspartase from Bacillus sp. YM55-1: structure-based exploration of functional sites in the aspartase family.

The crystal structure of the thermostable aspartase from Bacillus sp. YM55-1 has been solved and refined for 2.5A resolution data with an R-factor of 22.1%. The present enzyme is a homotetramer with subunits composed of three domains. It exhibits no allosteric effects, in contrast to the Escherichia coli aspartase, which is activated by divalent metal cation and L-aspartate, but is four-times more active than the E.coli enzyme. The overall folding of the present enzyme subunit is similar to those of the E.coli aspartase and the E.coli fumarase C, both of which belong to the same superfamily as the present enzyme. A local structural comparison of these three enzymes revealed seven structurally different regions. Five of the regions were located around putative functional sites, suggesting the involvement of these regions into the functions characteristic of the enzymes. Of these regions, the region of Gln96-Gly100 is proposed as a part of the recognition site of the alpha-amino group in L-aspartate for aspartase and the hydroxyl group in L-malate for fumarase. The region of Gln315-Gly323 is a flexible loop with a well-conserved sequence that is suggested to be involved in the catalytic reaction. The region of Lys123-Lys128 corresponds to a part of the putative activator-binding site in the E.coli fumarase C. The region in the Bacillus aspartase, however, adopts a main-chain conformation that prevents the activator binding. The regions of Gly228-Glu241 and Val265-Asp272, which form a part of the active-site wall, are suggested to be involved in the allosteric activation of the E.coli aspartase by the binding of the metal ion and the activator. Moreover, an increase in the numbers of intersubunit hydrogen bonds and salt-bridges is observed in the Bacillus aspartase relative to those of the E.coli enzyme, implying a contribution to the thermostability of the present aspartase.

Allosteric Site↗
PubMed1999

Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists.

Members of the BCL2 family of proteins are key regulators of programmed cell death, acting either as apoptotic agonists or antagonists. Here we describe the solution structure of BID, presenting the structure of a proapoptotic BCL2 family member. An analysis of sequence/structure of BCL2 family members allows us to define a structural superfamily, which has implications for general mechanisms for regulating proapoptotic activity. It appears two criteria must be met for proapoptotic function within the BCL2 family: targeting of molecules to intracellular membranes, and exposure of the BH3 death domain. BID's activity is regulated by a Caspase 8-mediated cleavage event, exposing the BH3 domain and significantly changing the surface charge and hydrophobicity, resulting in a change of cellular localization.

Amino Acid Sequence↗
PubMed2002

Structures of HO(2)-Co(III)bleomycin A(2) bound to d(GAGCTC)(2) and d(GGAAGCTTCC)(2): structure-reactivity relationships of Co and Fe bleomycins.

HO(2)-Co(III)bleomycin is a model for HO(2)-Fe(III)bleomycin, which initiates single and double strand cleavage of DNA. In order to enlarge the understanding of its structure and reactivity, three-dimensional structures of HO(2)-Co(III)bleomycin bound to two DNA oligomers, d(GAGCTC)(2) (I) and d(GGAAGCTTCC)(2) (II), that have 5'-GC-3' binding sites, have been determined by nuclear magnetic resonance (NMR) methods. Besides previously recognized determinants of binding selectivity, a probable hydrogen bond was detected between the pyrimidinyl acetamido NH(2) and the carbonyl of cytosine base paired to G at the recognition site. Another hydrogen bond between the NH of the dimethylsulfonium R group and N7 of guanine opposite cytosine at the GC site may contribute to specification of the pyrimidine. Substitution of G with inosine shifted HO(2)-Co(III)Blm A(2)[bond]I and Fe(III)Blm[bond]I into fast exchange on the NMR time scale, supporting the role of the 2-amino group in site specification for each molecule. The conformationally stable metal-domain linker established a close-packed adduct with the minor groove in which the hydroperoxide ligand occupies a sterically constrained pocket that is isolated from the solvent. The hydroperoxide group is directed toward one of the two cytosine H4' hydrogens but is sterically blocked from access to the other by the drug. These findings enlarge the structural understanding of selective binding of Co(III)/Fe(III)Blm species at G-pyrimidine sites. They also rationalize the instability of a number of ligands bound to Co(III)/Fe(III)Blm at specific binding sequences and the relative unreactivity of Fe(III)Blm[bond]I with ascorbate as well as its lack of interaction with spin labels.

Antibiotics, Antineoplastic↗
PubMed1998

Theoretical evidence of the existence of a diazafulvene intermediate in the reaction pathway of imidazoleglycerol phosphate dehydratase: design of a novel and potent heterocycle structure for the inhibitor on the basis of the electronic structure-activity relationship study.

The reaction mechanism of imidazoleglycerol phosphate dehydratase has not yet been clearly revealed. Structural comparison between inhibitors and the substrate IGP implicates that the reaction involves a diazafulvene intermediate. Here, we present evidence to support this hypothesis by investigating the electronic structure-enzyme inhibitory activity relationship on inhibitors with different heterocycles using 6-31G** level theory of the ab initio molecular orbital method. The calculation results showed that potent inhibitors can be distinguished from weak ones by the atomic charge density and by the energy levels of the highest occupied lone-pair orbital on the nitrogen atoms in the heterocycles. Furthermore, very good correlations (r2=0.8-0.9) were found between the charge density on the nitrogen atom and the inhibitory activity. It was also revealed that the diazafulvene is electronically similar to the potent inhibitors. Thus, these results strongly suggest the existence of the diazafulvene as an intermediate possessing tight-binding affinity to the enzyme. Based on the electronic structural similarity between the potent inhibitors and the proposed intermediate, a novel heterocycle was designed and predicted its inhibitory activity prior to the synthesis. Then, activity of synthesized inhibitors showed excellent agreement with this prediction. Hence, from the theoretical studies and experimental results, we conclude to obtain evidence of the hypothesis that the enzyme reaction proceeds via the diazafulvene intermediate.

Drug Design↗
PubMed1999

The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1.

The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.

Amino Acid Motifs↗
PubMed2006

Propagating structural perturbation inside bacteriorhodopsin: crystal structures of the M state and the D96A and T46V mutants.

The X-ray diffraction structure of the non-illuminated D96A bacteriorhodopsin mutant reveals structural changes as far away as 15 A from residue 96, at the retinal, Trp-182, Ala-215, and waters 501, 402, and 401. The Asp-to-Ala side-chain replacement breaks its hydrogen bond with Thr-46, and the resulting separation of the cytoplasmic ends of helices B and C is communicated to the retinal region through a chain of covalent and hydrogen bonds. The unexpected long-range consequences of the D96A mutation include breaking the hydrogen bond between O of Ala-215 and water 501 and the formation of a new hydrogen bond between water molecules 401 and 402 in the extracellular region. Because in the T46V mutant a new water molecule appears at Asp-96 and its hydrogen-bond to Ile-45 replaces Thr-46 as its link to helix B, the separation of helices B and C is smaller than that in D96A, and there are no atomic displacements elsewhere in the protein. Propagation of conformational changes along the chain between the retinal and Thr-46 had been observed earlier in the crystal structures of the D96N and E204Q mutants but in the trapped M state. Consistent with the perturbation of the retinal region in D96A, little change of the Thr-46 region occurs between the non-illuminated and M states of this mutant. It appears that a local perturbation can propagate along a track in both directions between the retinal and the Asp-96/Thr-46 pair, either from photoisomerization of the retinal in the wild-type protein in one case or from the D96A mutation in the other.

Amino Acid Substitution↗
PubMed1998

Structural symmetry: the three-dimensional structure of Haemophilus influenzae diaminopimelate epimerase.

The Haemophilus influenzae diaminopimelate epimerase was cloned, expressed, purified, and crystallized in the C2221 space group (a = 102.1 A, b = 115.4 A, c = 66.3 A, alpha = beta = gamma = 90 degrees). The three-dimensional structure was solved to 2.7 A using a single Pt derivative and the Se-Met-substituted enzyme to a conventional R factor of 19.0% (Rfree = 24.2%). The 274 amino acid enzyme consists of two structurally homologous domains, each containing eight beta-strands and two alpha-helices. Diaminopimelate epimerase is a representative of the PLP-independent amino acid racemases, for which no structure has yet been determined and substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. Cys73 of the amino terminal domain is found in disulfide linkage, at the domain interface, with Cys217 of the carboxy terminal domain, and we suggest that these two cysteine residues in the reduced, active enzyme function as the acid and base in the mechanism.

Amino Acid Isomerases↗
PubMed2004

Structural basis for antioxidant activity of trans-resveratrol: ab initio calculations and crystal and molecular structure.

From the experimental crystal structure and ab initio calculations on resveratrol and its derivatives, structural features of mechanistic importance are described. The molecular structure reveals the relative coplanarity of the trans-stilbene skeleton, and the molecular packing in the solid state shows an extensive hydrogen bond network that elucidates the flip-flop motion of the three hydroxyl groups that alternately form and break H bonds with each of the neighboring phenolic oxygens. The dynamic behavior provoked by the alternation of hydrogen bond formation and breaking can result in the ready mobility of up to three hydrogen atoms per resveratrol molecule that can be transferred to reactive oxidants that are rich in electron density. In addition, theoretical studies confirm the planarity of resveratrol as well as for half of the molecule of a condensation dimeric derivative of resveratrol, trans-sigma-viniferin. Furthermore, these studies show the p-4'-OH group to be more acidic compared to the other two m-OH groups. These features correlate with the biological activity of resveratrol as an antioxidant and support earlier studies showing H-atom transfer to be the dominant mechanism by which phenolic antioxidants intercept free radicals.

Antioxidants↗
PubMed1998

Comprehensive study on structure-activity relationships of rifamycins: discussion of molecular and crystal structure and spectroscopic and thermochemical properties of rifamycin O.

The mechanism of action of rifamycins against bacterial DNA-dependent RNA polymerase has been explained on the basis of the spatial arrangement of four oxygens which can form hydrogen bonds with the enzyme. Structural descriptors are derived from X-ray diffraction crystal structures of 25 active and nonactive rifamycins. Principal component analysis is used to find the combination of structural parameters which better discriminate between active and nonactive rifamycins. Two possible mechanisms of molecular rearrangement are described which can convert nonactive into active conformations. The energy involved for conformational rearrangements is studied by molecular modeling techniques. Methyl C34 is found to play a key role for determining the geometry of the pharmacophore. Rifamycin O, reported to be active, is obtained by oxidation of rifamycin B and is studied by X-ray single-crystal diffractometry, by solution IR and NMR spectroscopy, and by thermal analysis. Surprisingly the oxidation process is totally stereospecific, and an explanation is given based on solution spectroscopic evidence. The conformation found in the solid state is typical of nonactive compounds, and molecular mechanics calculations show that a molecular rearrangement to the active conformation would require about 15 kcal/mol. Thermal analysis confirms that rifamycin O has a sterically constrained conformation. Therefore, it is likely that the antibiotic activity of rifamycin O is due either to chemical modification prior to reaching the enzyme or to conformational activation.

Calorimetry, Differential Scanning↗
PubMed1999

Structure-immunosuppressive activity relationships of new analogues of 15-deoxyspergualin. 1. Structural modifications of the hydroxyglycine moiety.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Using the general concept of bioisosteric replacement, variations of the hydroxyglycine central "C" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was discovered as the first example of a new series of potent immunosuppressive agents encompassing a retro-amide bond linked to the hexyl-guanidino moiety. Structure-activity relationships of this series were studied by synthesizing compounds 13g-i and 13k-s. Variation of the "right-amide" of 13a led to the urea 19a and the carbamates 23 and 27a which proved to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent derivative, being slightly more active than DSG in a heart allotransplantation model in rats. Due to the absence of chiral center in its structure and to its improved chemical stability compared to DSG, 27a was selected as a candidate for clinical evaluation.

Animals↗