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Z Dauter

Publications and source records attributed to Z Dauter.

At least 109 records · Page 6Linked to original sources
PubMed1994

The atomic structure of Carnation Mottle Virus capsid protein.

The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determined at 3.2 A resolution by the method of molecular replacement. Three-dimensional data were collected from a small number of crystals (sp.g. I23, a = 382.6 A) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non-hydrogen atoms performed by the program XPLOR, has led to an R-factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the beta-annulus around the 3-fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary X-ray analysis of leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities. LIF is a glycoprotein secreted by a number of different cell types in vitro. It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin-IP and EGF. Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA. The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukin-6 (IL-6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM). Here, we report the crystallization of the non-glycosylated human-like LIF expressed in E. coli. The present crystals diffract to 2.0 A using synchrotron radiation. They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 A, b = 45.3 A, c = 77.7 A and beta = 112.3 degrees.

Cloning, Molecular↗
PubMed1993

Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides.

BACKGROUND: The trefoil peptides are a rapidly growing family of peptides, mainly found in the gastrointestinal tract. There is circumstantial evidence that they stabilize the mucus layer, and may affect the rate of healing of the mucosal epithelium. RESULTS: We have determined the structure of porcine pancreatic spasmolytic polypeptide (PSP) to 2.5 A resolution. The polypeptide contains two trefoil domains. The domain structure is compact, and is composed of a central short antiparallel beta-sheet with one short helix above and one below it. This is a novel motif. The two domains are related by two-fold symmetry, and each domain contains a cleft. CONCLUSIONS: The cleft within each domain could accommodate a polysaccharide chain, and may therefore be responsible for binding mucin glycoproteins. We suggest that PSP may cross-link glycoproteins, explaining its ability to stabilize the mucus layer.

Amino Acid Sequence↗
PubMed1993

Structure and function of endoglucanase V.

Cellulose is the major polysaccharide component of plant cell walls and is the most abundant organic compound on the planet. A number of bacterial and fungal organisms can use cellulose as a food source, possessing cellulases (cellobiohydrolases and endoglucanases) that can catalyse the hydrolysis of the beta-(1,4) glycosidic bonds. They can be classified into seven distinct families. The three-dimensional structures of members of two of these families are known. Here we report the structure of a third cellulase, endoglucanase V, whose sequence is not represented in any of the above families. The enzyme is structurally distinct from the previously determined cellulases but is similar to a recently characterized plant defence protein. The active site region resembles that of lysozyme, despite the lack of structural similarity between these two enzymes.

Binding Sites↗
PubMed1993

Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate.

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Binding Sites↗
PubMed1993

Crystallographic analyses of an active HIV-1 ribonuclease H domain show structural features that distinguish it from the inactive form.

. An active recombinant preparation of the carboxy-terminal ribonuclease H (RNase H) domain of HIV-I reverse transcriptase has produced crystals of several different forms, including a trigonal prism form (P3(1); a = b = 52.03, c = 113.9 A with two molecules per asymmetric unit) and a hexagonal tablet form (P6(2)22 or P6(4)22; a = b = 93.5, c = 74.1 A with one molecule per asymmetric unit). The former appears to be isomorphous with crystals of a similar, but inactive, version of the enzyme that was used for a prior crystal structure determination [Davies, Hostomska, Hostomsky, Jordan & Matthews (1991). Science, 252, 88-95]. We have also obtained a structure solution for this crystal form and have refined it with 2.8 A resolution data (R = 0.216). We report here details of our crystallization studies and some initial structural results that verify that the preparation of active HIV-1 RNase H yields a protein that is not just enzymatically, but also structurally, distinguishable from the inactive form. Evidence suggests that region 538-542, which may be involved in the catalytic site and which is disordered in both molecules in the prior structure determination, is ordered in the crystal structure of the active enzyme, although the ordering may include more than one conformation for this loop. It should also be noted that, in the crystal structure of the trigonal form, RNase H monomers associate to form noncrystallographic twofold-symmetric dimers by fusing five-stranded mixed beta sheets into a single ten-stranded dimerwide sheet, an assembly that was not remarked upon by previous investigators.

Journal Article↗
PubMed1993

Evaluation of mutagenesis for epitope mapping. Structure of an antibody-protein antigen complex.

The location and description of epitopes on proteins describe the basis of immunological specificity. The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined. This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R. E., Delbaere, L. T. J., Lee, J. S., and Waygood, E. B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4877-4881), with those from x-ray analysis. There are 14 amino acid residues of E. coli HPr that interact with the Jel42 antigen-binding site. Nine of these were correctly assigned by the mutagenesis studies. Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent. Four residues were incorrectly assigned to the epitope. These residues were located adjacent to epitope residues that were likely perturbed by these mutations. This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incorrect assignments.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of tyrosine phenol-lyase.

Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacter freundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH4-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5'-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5'-phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-A resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the crystallographic 2-fold symmetry axis of the crystal which belongs to the space group P2(1)2(1)2 with a = 76.0 A, b = 138.3 A, and c = 93.5 A. Each subunit comprises 14 alpha-helices and 16 beta-strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5'-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5'-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.

Amino Acid Sequence↗
PubMed1993

Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin.

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 multimer structure, in which the B pentamer has a membrane binding function and the A subunit is needed for enzymatic activity. The LT crystal structure has been solved using a combination of multiple isomorphous replacement, fivefold averaging and molecular dynamics refinement. Phase combination using all these sources of phase information was of crucial importance for the chain tracing. The structure has now been refined to 1.95 A resolution, resulting in a model containing 6035 protein atoms and 293 solvent molecules with a crystallographic R-factor of 18.2% and good stereochemistry. The B subunits are arranged as a highly stable pentamer with a donut shape. Each subunit takes part in approximately 30 inter-subunit hydrogen bonds and six salt bridges with its two neighbors, whilst burying a large surface area. The A subunit has higher temperature factors and less well-defined secondary structure than the B subunits. It interacts with the B pentamer mainly via the C-terminal A2 fragment, which runs through the highly charged central pore of the B subunits. The pore contains at least 66 water molecules, which fill the space left by the A2 fragment. A detailed analysis of the contacts between A and B subunits showed that most specific contacts occur at the entrance of the central pore of the B pentamer, while the contacts within the pore are mainly hydrophobic and water mediated, with the exception of two salt bridges. Only a few contacts exist between the A1 fragment and the B pentamer, showing that the A2 fragment functions as a "linker" of the A and B parts of the protein. Interacting with the A subunit by the B subunits does not cause large deviations from a common B subunit structure, and the 5-fold symmetry is well maintained. A potential NAD(+)-binding site is located in an elongated crevice at the interface of two small sheets in the A1 fragment. At the back of this crevice the functionally important Arg7 makes a hydrogen bond connecting two strands, which seems to be conserved across the ADP-ribosylating toxin family. The putative catalytic residue (A1:Glu112) is located nearby, close to a very hydrophobic region, which packs two loops together. This hydrophobic region may be important for catalysis and membrane translocation.

Amino Acid Sequence↗
PubMed1993

Structures of the lectin IV of Griffonia simplicifolia and its complex with the Lewis b human blood group determinant at 2.0 A resolution.

The structures of the fourth lectin isolated from Griffonia simplicifolia (GS4) and its complex with the methyl-glycoside of the Lewis b human blood group determinant (Le(b)-OMe) are reported at high resolution. The native GS4 crystal is isomorphous with the complexed GS4 crystal. The space group is P4(2)2(1)2 with unit cell dimensions a = 78.9 A, c = 89.1 A with one subunit of the lectin (bound to 1 Le(b)-OMe in the complex) in the crystallographic asymmetric unit. The native GS4 structure was solved by the molecular replacement technique and least-squares refined (PROLSQ and X-PLOR). The orientation of the Le(b)-OMe tetrasaccharide in the complex was established from a 2.8 A difference map with coefficients (Fcomplex--Fnative) and calculated phase angles from the native model. Both the final native and complex GS4 models consist of 1904 protein non-hydrogen atoms, one sulfate ion, one Ca ion, one Mn ion and three covalently-bound sugar residues N-linked to Asn18. In addition, the complex model has 47 Le(b)-OMe non-hydrogen atoms. The two structures have 135 water molecules in common in addition to eight and nine unique water molecules in the native and complex structures, respectively. The root-mean-square deviations from ideal bond distances and angles are 0.016 A, 3.2 degrees and 0.016 A, 3.0 degrees, for the native and complexed GS4, respectively. The R index for all unique data from 8 to 2.0 A is 0.187 for the native (19,204 reflections) and 0.181 for the complex (19,212 reflections). The tertiary structure of each subunit is similar to that of other leguminous lectins but the quaternary structure of the molecular dimer is different from that of any other lectin reported to date. The co-ordination about the Ca ion is pentagonal bipyramidal (with 1 long Ca(2+)-oxygen bond) and the co-ordination about the Mn ion is octahedral. Two conserved residues (Asp149 and Ser155) appear to be important because they are hydrogen-bonded to each other and to groups that co-ordinate the Mn ion. There are three cis-peptides in the polypeptide chain; two involve non-proline residues, one of which is homologous with other leguminous lectins and the other is unique to GS4. The two non-proline cis-peptides are located in the carbohydrate-binding site and are important for the specificity of the lectin. The molecular recognition of Le(b)-OMe by GS4 involves both polar and extensive non-polar interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1993

Complex of ribonuclease from Streptomyces aureofaciens with 2'-GMP at 1.7 A resolution.

The crystal structure of a complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with guanosine-2'-monophosphate (2'-GMP) has been refined against synchrotron data recorded from a single crystal using radiation from beamline X31 at EMBL, Hamburg, and an imaging plate scanner. The crystals are in space group P2(1)2(1)2(1) with cell dimensions a = 64.7, b = 78.8 and c = 39.1 A. The structure has two enzyme molecules in the asymmetric unit, complexed with 2'-GMP inhibitor with occupancies of 1 and 2/3 (different to the 3'-GMP complex crystal structure where only one of the two independent RNase Sa molecules binds nucleotide), 492 associated water molecules and one sulfate ion, and was refined using all data between 10.0 and 1.7 A to a final crystallographic R factor of 13.25%. Binding of the base to the enzyme confirms the basis for the guanine specificity but the structural results still do not provide direct evidence of the identity and role of the particular residues involved in the catalytic process. New native RNase Sa data to 1.8 A were recorded to provide a reference set measured under comparable experimental conditions. The crystals are in the same space group and have the same lattice as those of the 2'-GMP complex. The native structure with 423 water molecules was refined in a similar manner to the complex to a final R factor of 13.87%. 1.77 A resolution data were independently measured on a 2'-GMP complex crystal at UCLA using an R-AXIS II image plate scanner mounted on a conventional source. The cell dimensions were essentially the same as above. 2'-GMP was bound more fully to molecule A than to molecule B of the RNase Sa. The structure was refined to an R factor of 14.64% with 388 water molecules. This work follows on from the structure determination of native RNase Sa and its complex with 3'-GMP [Sevcik, Dodson & Dodson (1991). Acta Cryst. B47, 240-253].

Journal Article↗
PubMed1993

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 A resolution.

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.

Hydrolysis↗
PubMed1993

The application of direct methods and Patterson interpretation to high-resolution native protein data.

Conventional small-molecule methods of solving the phase problem from native data alone, without the use of heavy-atom derivatives, known fragment geometries or anomalous dispersion, have been tested on 0.9 A resolution data for two small proteins: rubredoxin, from Desulfovibrio vulgaris, and crambin. The presence of three disulfide bridges in crambin and an FeS(4) unit in rubredoxin enabled automated Patterson interpretation as well as direct methods to be tried. Although both structures were already well established, the known structures were not used in the phasing attempts, except for identifying successful solutions. Direct methods were not successful for crambin, although the correct phases were stable to phase refinement and gave figures of merit clearly superior to any obtained in the ca 500 000 random starting phase sets that were refined. It appears that the presence of an iron atom in rubredoxin reduces the scale of the search problem by many orders of magnitude, but at the cost of producing 'over-consistent' phase sets that overemphasize the iron atom and involve partial loss of enantiomorph information. However, about 1% of direct-methods trials were successful for rubredoxin, giving mean phase errors of about 56 degrees (for all E > 1.2) that could be reduced to about 20 degrees by standard E-Fourier recycling methods. Limiting the resolution of the data degraded the quality of the solutions and suggested that the limiting resolution for routine direct-methods solution of rubredoxin is about 1.2 A. With the 0.9 A data, automated Patterson interpretation convincingly finds the three disulfide bridges in crambin and the FeS(4) unit in rubredoxin, and in both cases E-Fourier recycling starting from these 'heavier' atoms yields almost the complete structure. Whereas crambin could only be solved in this way at very high resolution, rubredoxin could be solved by Patterson interpretation down to 1.6 A. These results emphasize the benefits of collecting protein data to the highest possible resolution, and indicate that when a few 'heavier' atoms are present, it may prove possible in favorable cases to solve the phase problem from a single native data set collected to 'atomic resolution'.

Journal Article↗
PubMed1993

Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 A resolution: clues for a mechanism for allosteric regulation.

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

Allosteric Regulation↗
PubMed1993

A monoclonal antibody Fab fragment crystallized with and without a peptide epitope from HIV.

The Fab fragment of CB 4-1, a monoclonal murine antibody against HIV protein p24, has been produced. It forms a complex with a synthetic antigen, an epitope of p24 made up of 11 amino acids, with the binding constant Kd = 3.6 x 10(-9) M. Crystals of hexagonal and orthorhombic space group has been obtained by cocrystallization of the Fab with the epitope and crystallization without the epitope, respectively. In either case, the crystals are suitable for X-ray structural analysis. Crystals of the Fab fragment cocrystallized with the peptide have the space group P 6(3)22 with cell dimensions of a = b = 105 A, c = 297 A. Fab crystals without the epitope are in space group C 222 with cell dimensions a = 110.1 A, b = 110.2 A, c = 150.1 A.

Animals↗
PubMed1993

A new crystal form of tropomyosin.

Tropomyosin crystals with a new morphology have been obtained from lobster tail muscle tropomyosin from which 11 residues at the carboxyl-terminus have been proteolytically removed to avoid head-to-tail polymerization. In contrast to the conventional Bailey crystal form in which the elongated tropomyosin molecules form a mesh, in the present crystals the molecules are packed side-to-side with the long axes parallel to the c-axis of the crystal. The unit cell is tetragonal with a = b = 109 A, c = 509 A, and the symmetry is either P4(1)2(1)2 or P4(3)2(1)2, with 4(1)(4(3)) helical axes parallel to the c-axis. This suggests that a group of molecules surrounding a local 4(1)(4(3)) axis is regarded as the building unit of the crystal. It is likely that the unit cell contains eight molecules with one molecule per asymmetric unit.

Amino Acid Sequence↗
PubMed1993

The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Amino Acid Sequence↗