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At least 793 records · Page 44Linked to original sources
PubMed1999

Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability.

The crystal structures of highly thermostable xylose isomerases from Thermus thermophilus (TthXI) and Thermus caldophilus (TcaXI), both with the optimum reaction temperature of 90 degrees C, have been determined by X-ray crystallography. The model of TcaXI has been refined to an R-factor of 17.8 % for data extending to 2.3 A and that of TthXI to 17.1 % for data extending to 2.2 A. The tetrameric arrangement of subunits characterized by the 222-symmetry and the tertiary fold of each subunit in both TcaXI and TthXI are basically the same as in other xylose isomerases. Each monomer is composed of two domains. Domain I (residues 1 to 321) folds into the (beta/alpha)8-barrel. Domain II (residues 322 to 387), lacking beta-strands, makes extensive contacts with domain I of an adjacent subunit. Each monomer of TcaXI contains ten beta-strands, 15 alpha-helices, and six 310-helices, while that of TthXI contains ten beta-strands, 16 alpha-helices, and five 310-helices. Although the electron density does not indicate the presence of bound metal ions in the present models of both TcaXI and TthXI, the active site residues show the conserved structural features. In order to understand the structural basis for thermostability of these enzymes, their structures have been compared with less thermostable XIs from Arthrobacter B3728 and Actinoplanes missouriensis (AXI and AmiXI), with the optimum reaction temperatures of 80 degrees C and 75 degrees C, respectively. Analyses of various factors that may affect protein thermostability indicate that the possible structural determinants of the enhanced thermostability of TcaXI/TthXI over AXI/AmiXI are (i) an increase in ion pairs and ion-pair networks, (ii) a decrease in the large inter-subunit cavities, (iii) a removal of potential deamidation/isoaspartate formation sites, and (iv) a shortened loop.

Aldose-Ketose Isomerases↗
PubMed2001

Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism.

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

Amino Acid Substitution↗
PubMed2002

Crystal structures of the sugar complexes of Streptomyces olivaceoviridis E-86 xylanase: sugar binding structure of the family 13 carbohydrate binding module.

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.

Binding Sites↗
PubMed1992

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: effect of the hydrophobic chain length of the surfactants on the structural changes.

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion of alpha-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, the alpha-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.

Anions↗
PubMed1990

The structure of the carboxyl terminus of the p21 protein. Structural relationship to the nucleotide-binding/transforming regions of the protein.

The carboxyl-terminal region of the ras oncogene-encoded p21 protein is critical to the protein's function, since membrane binding through the C-terminus is necessary for its cellular activity. X-ray crystal structures for truncated p21 proteins are available, but none of these include the C-terminal region of the protein (from residues 172-189). Using conformational energy analysis, we determined the preferred three-dimensional structures for this C-terminal octadecapeptide of the H-ras oncogene p21 protein and generated these structures onto the crystal structure of the remainder of the protein. The results indicate that, like other membrane-associated proteins, the membrane-binding C-terminus of p21 assumes a helical hairpin conformation. In several low-energy orientations, the C-terminal structure is in close proximity to other critical locales of p21. These include the central transforming region (around Gln 61) and the amino terminal transforming region (around Gly 12), indicating that extracellular signals can be transduced through the C-terminal helical hairpin to the effector regions of the protein. This finding is consistent with the results of recent genetic experiments.

Amino Acid Sequence↗
PubMed2006

Comparison of a homology model and the crystallographic structure of human 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) in a structure-based identification of inhibitors.

Human 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) catalyzes the interconversion of cortisone into active cortisol. 11betaHSD1 inhibition is a tempting target for the treatment of a host of human disorders that might benefit from blockade of glucocorticoid action, such as obesity, metabolic syndrome, and diabetes type 2. Here, we report an in silico screening study aimed at identifying new selective inhibitors of human 11betaHSD1 enzyme. In the first step, homology modeling was employed to build the 3D structure of 11betaHSD1. Further, molecular docking was used to validate the predicted model by showing that it was able to discriminate between known 11betaHSD1 inhibitors or substrates and non-inhibitors. The homology model was found to reproduce closely the crystal structure that became publicly available in the final stages of this work. Finally, we carried out structure-based virtual screening experiments on both the homology model and the crystallographic structure with a database of 114,000 natural molecules. Among these, 15 molecules were consistently selected as inhibitors based on both the model and crystal structures of the enzyme, implying a good quality for the homology model. Among these putative 11betaHSD1 inhibitors, two were flavonone derivatives that have already been shown to be potent inhibitors of the enzyme.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1991

Structure-activity relationships in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds in rats.

We investigated the structure-activity relationship in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds. For this purpose, we synthesized various compounds structurally analogous to clotrimazole and injected them into rats at a fixed dose of 0.2 mmol/kg. We found that the chlorine atom in clotrimazole was not necessary for the induction of cytochrome P450. The imidazole moiety of clotrimazole, however, was a very important structural component for the induction of cytochrome P450; triazole, but not pyrrole, could be substituted for this moiety. 1-Tritylimidazole, 1-diphenylmethylimidazole and 1-benzylimidazole were also found to be inducers of cytochrome P450, but, to a somewhat lesser extent, with a decreasing number of substituted phenyl groups. Thus, 1-benzylimidazole was a minimum structurally active unit for inducing cytochrome P450. In addition, 4-diphenylmethylpyridine and 4-benzylpyridine also induced cytochrome P450 to extents similar to those induced by the corresponding imidazole derivatives, but 4-benzylpiperidine lacked this effect. When the methylene unit of clotrimazole-related compounds was introduced by a hydroxy or amino group instead of imidazole, there was a less extensive increase in cytochrome P450 content. This inducing effect was lost completely by the lack of an imidazole moiety and imidazole itself. 1-Phenylethylimidazole and 1-benzylimidazole induced cytochrome P450 to a similar extent. All of these findings suggest that 1-substituted heteroaromatic compounds having two or more nitrogen atoms are likely to be required for inducing cytochrome P450. Immunoblot analysis revealed that clotrimazole and other various inducers found in this study increased cytochrome P450b/e content. These results could provide information on the effects of drugs and chemicals on cytochrome P450 induction.

Animals↗
PubMed1987

The crystal structures of octyl alpha-D-glucopyranoside monohydrate and hemihydrate: mesogenic structures with interdigitizing alkyl chains.

The crystal structure of octyl alpha-D-glucopyranoside monohydrate, C14H28O6.H2O, is monoclinic, C2, with Z = 4, a = 17.896(2), b = 5.154(1), c = 18.303(2) A, beta = 90.30(1) degrees. The hemihydrate, C14H28O6.0.5 H2O, is also monoclinic, C2, with Z = 4, a = 15.190(5), b = 5.136(3), c = 19.944(7), beta = 92.74(3) degrees. The crystal structures were solved using SHELXTL and refined to R values of 0.037 and 0.052 for 1224 and 1231 observed structure amplitudes, respectively. The crystal structures have bilayer head-to-head molecular packing with interdigitizing alkyl chains similar to those observed in other long-chain alkyl pyranosides. The carbohydrate moieties are hydrogen-bonded in infinite chains which exclude the ring and glycosidic oxygen atoms. Both crystal structures transform to a smectic A liquid crystal at 72.3 degrees, which has a clearing point at 116.5 degrees.

Carbohydrate Conformation↗
PubMed1994

The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop.

SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases. The crystal structure of the SH3 domain of Csk (c-Src specific tyrosine kinase) has been refined at a resolution of 2.5 A, with an R-factor of 22.4%. The structure is very similar to the FynSH3 crystal structure. When comparing CskSH3 and FynSH3 it is seen that the structural and charge differences of the RT-Src loop and the n-Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains. The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.

Amino Acid Sequence↗
PubMed1992

Structure-derived hydrophobic potential. Hydrophobic potential derived from X-ray structures of globular proteins is able to identify native folds.

We present a model for the hydrophobic interaction in globular proteins that is based entirely on an analysis of known X-ray structures. This structure-derived hydrophobic force is identified as the strongest among the non-covalent interactions that stabilize native folds. The functional form of the hydrophobic interaction is found to be linear, corresponding to a constant force along the observable distance range (5 to 70 A). The parameters of the hydrophobic amino acid pair potentials yield a structure-derived hydrophobicity scale that correlates strongly with scales derived by a variety of complementary approaches. We demonstrate that the structure-derived hydrophobic interaction alone is able to distinguish a substantial number of native conformations from a large pool of misfolded structures.

Amino Acid Sequence↗
PubMed2006

Quantitative structure-activity-activity and quantitative structure-activity investigations of human and rodent toxicity.

Acute toxicity in different biological systems, including humans and rodents in vivo, and human and rodent cell lines in vitro, was investigated. The data were taken from the MEIC (Multicentre Evaluation of In Vitro Cytotoxicity) programme. Quantitative structure-activity-activity relationship (QSAAR) models were developed for the in vivo human and rodent toxicity including a combination of toxicity endpoint and structural descriptors as predictor variables. The human peak blood/serum LC(50) concentrations were most strongly related to human liver cell toxicity, while the in vivo oral human lethal doses were most closely related to the in vivo rodent LD(50) values. The QSAARs included structural descriptors encoding electronic/reactivity properties, presence of H-bond donors, compound aromaticity, and size/shape properties. Quantitative structure-activity relationships (QSARs) were derived by using structural descriptors accounting for molecular hydrophobicity, size and shape, and electronic properties. These models have the potential to provide useful insights in the development of non-animal (in vitro and in silico) methods for predicting human and mammalian toxicity.

Animals↗
PubMed2004

Structure of human Ki67 FHA domain and its binding to a phosphoprotein fragment from hNIFK reveal unique recognition sites and new views to the structural basis of FHA domain functions.

Recent studies by use of short phosphopeptides showed that forkhead-associated (FHA) domains recognize pTXX(D/I/L) motifs. Solution structures and crystal structures of several different FHA domains and their complexes with short phosphopeptides have been reported by several groups. We now report the solution structure of the FHA domain of human Ki67, a large nuclear protein associated with the cell-cycle. Using fragments of its binding partner hNIFK, we show that Ki67-hNIFK binding involves ca 44 residues without a pTXX(D/I/L) motif. The pThr site of hNIFK recognized by Ki67 FHA is pThr234-Pro235, a motif also recognized by the proline isomerase Pin1. Heteronuclear single quantum coherence (HSQC) NMR was then used to map out the binding surface, and structural analyses were used to identify key binding residues of Ki67 FHA. The results represent the first structural characterization of the complex of an FHA domain with a biologically relevant target protein fragment. Detailed analyses of the results led us to propose that three major factors control the interaction of FHA with its target protein: the pT residue, +1 to +3 residues, and an extended binding surface, and that variation in the three factors is the likely cause of the great diversity in the function and specificity of FHA domains from different sources.

Binding Sites↗
PubMed2006

Structural basis of the nickel response in Helicobacter pylori: crystal structures of HpNikR in Apo and nickel-bound states.

The survival of Helicobacter pylori in the human stomach critically relies on the availability and use of nickel, an absolute cofactor of the important virulence determinant urease. Nickel-responsive gene regulation is mediated by HpNikR, a protein belonging to the ribbon-helix-helix family of transcriptional regulators. Unlike its homologues, HpNikR acts as both a repressor and an activator within an acid adaptation cascade. We report the crystal structure of the full-length HpNikR in a nickel-free conformation and two nickel-bound structures obtained in different conditions: Ni1-HpNikR and Ni2-HpNikR. Apo-HpNikR shows the same global fold as its bacterial homologues although with an unusual closed trans-conformation and asymmetrical quaternary arrangement. The structure of Ni1-HpNikR in the presence of nickel has two different sides, one showing nickel binding similar to that of known NikRs and the other reflecting an intermediate state. The structure of Ni2-HpNikR obtained using a shorter exposure to nickel provides another snapshot of the nickel incorporation. Altogether, the three structures have allowed us to determine the route for nickel within HpNikR and reveal the cooperativity between the tetramerization domain and the DNA-binding domain. Experiments using point mutations of HpnikR residues involved in nickel internalisation confirm that these residues are critical for HpNikR functions in vivo.

Bacterial Proteins↗
PubMed2003

Deacylated pulmonary surfactant protein SP-C transforms from alpha-helical to amyloid fibril structure via a pH-dependent mechanism: an infrared structural investigation.

Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, alpha-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the alpha-helix form of SP-C is metastable, and under certain circumstances may transform from an alpha-helix to a beta-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an alpha-helical to a beta-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is alpha-helical in nature, but converts to an amyloid fibril structure composed of short beta-strands or beta-hairpins at neutral pH. The alpha-helix structure of dSP-C is fully recoverable from the amyloid beta-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril beta-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated alpha-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel.

Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.

Amino Acid Sequence↗
PubMed2003

Structural and mechanistic insight from high resolution structures of archaeal rhodopsins.

Lipidic cubic phase-grown crystals yielded high resolution structures of a number of archaeal retinal proteins, the molecular mechanisms of which are being revealed as structures of photocycle intermediates become available. The structural basis for bacteriorhodopsin's mechanism of proton pumping is discussed, revealing a well-synchronized sequence of molecular events. Comparison with the high resolution structures of the halide pump halorhodopsin, as well as with the receptor sensory rhodopsin II, illustrates how small and localized structural changes result in functional divergence. Fundamental principles of energy transduction and sensory reception in the archaeal rhodopsins, which may have relevance to other systems, are discussed.

Archaeal Proteins↗
PubMed2002

Structural differences in the NOE-derived structure of G-T mismatched DNA relative to normal DNA are correlated with differences in (13)C relaxation-based internal dynamics.

Detailed description of the characteristics of mismatched DNA that are distinct from normal DNA is vital to the understanding of how mismatch repair proteins are able to recognize and repair these DNA lesions. To this end, we have used nuclear Overhauser effect spectroscopy (NOESY)-based distance restraints and (13)C relaxation measurements to solve the solution structures and measure some of the internal dynamics of the G-T mismatched DNA oligomer d(CCATGCGTGG)(2) (GT) and its parent DNA sequence d(CCACGCGTGG)(2) (GC). In GT, the mismatched G7 is structurally perturbed much more than the mismatched T4 relative to their corresponding bases in GC. The degree of G7 displacement differs from previous high-resolution structures of G-T mismatch-containing B-DNA, suggesting a dependence of G-T mismatch-induced structural perturbation on sequence context. The internal dynamics of GC and GT differ on multiple timescales. The mismatched G7 of GT contains spins that decrease significantly in order in GT compared to GC, while spins in C6, T8, and A3 have significantly higher order in GT compared to GC. Linear correlations between helical parameters of GC and GT and the order of C-1' and aromatic methine carbon atoms relate differences in internal dynamics to the structures quantitatively. The dynamic differences between the normal and mismatched DNA signify changes in local flexibility that may be exploited by the mismatch repair system to bind mismatched DNA preferentially while ignoring normal DNA.

Base Pair Mismatch↗
PubMed2002

Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins.

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.

Amino Acid Sequence↗