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Biomedical subjects

R Huber

Publications and source records attributed to R Huber.

At least 307 records · Page 17Linked to original sources
PubMed1996

Small structural changes account for the high thermostability of 1[4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

BACKGROUND: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest. Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities. The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases. Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified. RESULTS: The 1.75 A crystal structure of the extremely thermostable 1[4Fe-4S] ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability. CONCLUSIONS: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure. Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins. These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds. Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone. The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm.

Amino Acid Sequence↗
PubMed1996

Enzyme flexibility, solvent and 'weak' interactions characterize thrombin-ligand interactions: implications for drug design.

BACKGROUND: The explosive growth in the rate of X-ray determination of protein structures is fuelled largely by the expectation that structural information will be useful for pharmacological and biotechnological applications. For example, there have been intensive efforts to develop orally administrable antithrombotic drugs using information about the crystal structures of blood coagulation factors, including thrombin. Most of the low molecular weight thrombin inhibitors studied so far are based on arginine and benzamidine. We sought to expand the database of information on thrombin-inhibitor binding by studying new classes of inhibitors. RESULTS: We report the structures of three new inhibitors complexed with thrombin, two based on 4-aminopyridine and one based on naphthamidine. We observe several geometry changes in the protein main chain and side chains which accompany inhibitor binding. The two inhibitors based on 4-aminopyridine bind in notably different ways: one forms a water-mediated hydrogen bond to the active site Ser195, the other induces a rotation of the Ser214-Trp215 peptide plane that is unprecedented in thrombin structures. These binding modes also differ in their 'weak' interactions, including CH-O hydrogen bonds and interactions between water molecules and aromatic pi-clouds. Induced-fit structural changes were also seen in the structure of the naphthamidine inhibitor complex. CONCLUSIONS: Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Ligand design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the significance of 'weak' interactions must be considered both in crystallographic refinement and in analysis of binding mechanisms.

4-Aminopyridine↗
PubMed1996

The ornithodorin-thrombin crystal structure, a key to the TAP enigma?

Ornithodorin, isolated from the blood sucking soft tick Ornithodoros moubata, is a potent (Ki = 10(-12) M) and highly selective thrombin inhibitor. Internal sequence homology indicates a two domain protein. Each domain resembles the Kunitz inhibitor basic pancreatic trypsin inhibitor (BPTI) and also the tick anticoagulant peptide (TAP) isolated from the same organism. The 3.1 A crystal structure of the ornithodorin-thrombin complex confirms that both domains of ornithodorin exhibit a distorted BPTI-like fold. The N-terminal portion and the C-terminal helix of each domain are structurally very similar to BPTI, whereas the regions corresponding to the binding loop of BPTI adopt different conformations. Neither of the two 'reactive site loops' of ornithodorin contacts the protease in the ornithodorin-thrombin complex. Instead, the N-terminal residues of ornithodorin bind to the active site of thrombin, reminiscent of the thrombin-hirudin interaction. The C-terminal domain binds at the fibrinogen recognition exosite. Molecular recognition of its target protease by this double-headed Kunitz-type inhibitor diverges considerably from other members of this intensely studied superfamily. The complex structure provides a model to explain the perplexing results of mutagenesis studies on the TAP-factor Xa interaction.

Amino Acid Sequence↗
PubMed1996

Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp.

Crystal structures of N-carbamoylsarcosine amidohydrolase (CSHase; EC 3.5.1.59) have been analyzed by X-ray diffraction methods with two different inhibitors bound to the active site at 2.28 A and 2.37 A resolution. The catalytic center of the enzyme could be identified on the basis of these structures. The four substrate binding sites are situated at the intersubunit interfaces of the compact dimers AB and CD of the homotetrameric enzyme. Both inhibitors inactivate the enzyme irreversibly through covalent binding of their aldehyde groups to the thiol group of the active-site cysteine residue Cys177. Within the identified substrate binding sites a number of residues from different subunits are involved in hydrogen bonding of the inhibitors. Two residues (Ala172 and Thr173) that form an unusual cis-peptide bond at the binding site are important components in fixing the examined inhibitors by hydrogen bonds. An electrochemical enzyme assay for CSHase was used to test the effect of inhibitors and substrate analogs on the enzyme's activity, revealing the high substrate specificity of CSHase. The intrinsic tryptophan fluorescence of CSHase increases strongly upon substrate and inhibitor binding. As most of the tryptophyl residues are located at the active sites, they are thus considerably affected by ligand binding. Fluorescence-detected stopped-flow measurements have been used to study the kinetics of glyoxylate and substrate binding to CSHase. Substrate and inhibitor binding could clearly be distinguished in the stopped-flow experiments. Inhibitor binding reveals at least three different elementary processes, whereas substrate binding is much faster and contains phases with different signs in amplitude.

Amidohydrolases↗
PubMed1996

Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

The operon encoding the periplasmic enzymes dimethyl sulfoxide reductase (DMSOR) and trimethylamine N-oxide reductase (TMAOR) from the purple, non-sulfur, photosynthetic bacterium Rhodobacter capsulatus was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences generated by automated Edman degradation and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/ TMAOR operon. The promoter consisted of an A + T-rich region followed by a -35 region, a putative ribosome binding site, and a leader sequence of 13 amino acid residues. The transcription terminator was a G + C-rich dyad sequence capable of forming a hairpin structure, which may act rho-independently. An optimized protein purification of the wild-type enzyme is also described, giving high yields (5 mg protein per liter of culture) and a specific activity of 30 units/mg. The molecular mass was determined by electrospray mass spectrometry to be 85,034 Da; from the deduced amino acid sequence the molecular mass of the apoenzyme was 85,033 Da.

Amino Acid Sequence↗
PubMed1996

Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 A resolution.

The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A. The crystals diffract beyond 1.8 A with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away. It might be involved in electron shuttling from the protein surface to the molybdenum center.

Amino Acid Sequence↗
PubMed1996

Crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications for selectivity.

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with two opposite specificities.

The crystal structure of human neutrophil cathepsin G, complexed with the peptidyl phosphonate inhibitor Suc-Val-Pro-PheP-(OPh)2, has been determined to a resolution of 1.8 A using Patterson search techniques. The cathepsin G structure shows the polypeptide fold characteristic of trypsin-like serine proteinases and is especially similar to rat mast cell proteinase II. Unique to cathepsin G, however, is the presence of Glu226 (chymotrypsinogen numbering), which is situated at the bottom of the S1 specificity pocket, dividing it into two compartments. For this reason, the benzyl side chain of the inhibitor PheP residue does not fully occupy the pocket but is, instead, located at its entrance. Its positively charged equatorial edge is involved in a favourable electrostatic interaction with the negatively charged carboxylate group of Glu226. Arrangement of this Glu226 carboxylate would also allow accommodation of a Lys side chain in this S1 pocket, in agreement with the recently observed cathepsin G preference for Lys and Phe at P1. The cathepsin G complex with the covalently bound phosphonate inhibitor mimics a tetrahedral substrate intermediate. A comparison of the Arg surface distributions of cathepsin G, leukocyte elastase and rat mast cell protease II shows no simple common recognition pattern for a mannose-6-phosphate receptor-independent targeting mechanism for sorting of these granular proteinases.

Amino Acid Sequence↗
PubMed1996

Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

Bacteriophage P22↗
PubMed1996

Site-directed mutagenesis and structural interpretation of the nidogen binding site of the laminin gamma1 chain.

A precise molecular map of the nidogen binding site of laminins was obtained by site-directed mutagenesis and structural analysis of the 56 residue LE module gamma1III4 of their gamma1 chain. This demonstrated the crucial importance of the sequence DPNAV (position 800-804) in the disulfide-bonded loop a, with major contributions made by all residues except P801. Different substitutions of these residues emphasized the essential role of the negative charge (D800) and carboxamide group (N802) as well as their spacings and hydrophobic contacts (V804) for interaction, and predict direct contacts of these three residues with a complementary binding region of nidogen. An inactivating A803-V substitution, however, may lead to a distorted loop structure. A lower but still significant contribution originates from the non-contiguous link/loop c sequence LKCIY (positions 815-819) which is spatially close to the loop a sequence. The link residues (L815 and K816) provide main chain hydrogen bonds to N806 and a side chain hydrogen bond to the V804 carbonyl and thus stabilize the conformation of loop a. The side chains of I818 and Y819 together with P842 from loop d form hydrophobic contacts that provide further stability but could possibly also participate in direct ligation. The nidogen binding epitope is therefore localized on a narrow ridge and has a length of approximately 17 angstroms. The data also indicate a strong conservation of the epitope in the laminin gamma1 chains of several invertebrates.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A.

Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.

Alanine↗
PubMed1996

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

Aldehyde Oxidoreductases↗
PubMed1996

The structure of recombinant human annexin VI in crystals and membrane-bound.

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.

Annexin A6↗
PubMed1996

Carbohydrate and protein-based inhibitors of porcine pancreatic alpha-amylase: structure analysis and comparison of their binding characteristics.

The crystal structures of porcine pancreatic alpha-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 angstroms, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface. V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder. This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments. A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.

Animals↗
PubMed1996

Structural and functional characterisation of the voltage sensor in the ion channel human annexin V.

The ion channel properties of human annexin V, a calcium- and phospholipid-binding protein of the annexin family, have been structurally and functionally investigated by analysing the mutant Glu112 -->Gly. Glu112 forms a salt bridge with Arg271 located in the interior of the hydrophilic pore of the molecule which is conserved within the annexin family. The crystal structures of the mutant and wild-type proteins are very similar and show only marginal conformational changes around the mutation site. Electron microscopic images show a conserved four-domain structure upon membrane binding as in the wild-type annexin V. The channel properties of the mutant are drastically changed, as the mutant has lost the voltage-dependent channel gating and the selectivity for calcium ions over monovalent cations. These results strongly support the hypothesis that the central, hydrophilic pore is the ion-conducting pathway.

Annexin A5↗
PubMed1996

Locating a local symmetry axis from patterson map cross vectors: application to crystal data from GroEl, GTP cyclohydrolase I and the proteosome.

The cross vectors of the native Patterson map are shown to exhibit non-crystallographic symmetry in the case of local axes parallel to one another. This information can be used to determine the translation component of such axes. A program is described to search for this cross vector, and is tested on low-resolution data from crystals of the tetradecameric GroEL molecule, the decameric GTP cyclohydrolase I and the tetradecameric proteosome. For GroEL, the function produces a packing arrangement optimal for sevenfold symmetry, and is in agreement with the dimensions of the molecule as given by electron microscopy data and the recently determined crystal structure. Positioning of local axes is confirmed by two high-resolution crystal structure analyses: the fivefold axis in cyclohydrolase I and the sevenfold axis in the proteosome. Implications for the location of heavy-atom positions are discussed for these two cases.

Journal Article↗
PubMed1996

The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.

Amino Acid Sequence↗