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PubMed2001

Primary structure, isoforms, and molecular modeling of a chitin-binding mistletoe lectin.

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1--48), the second is a heterodimer, containing one truncated (1--48) and one full-length chain (1--49), and the third is composed of two full length chains (1--49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.

Amino Acid Sequence↗
PubMed1994

Complete amino acid sequence and comparative molecular modelling of HPr from Streptococcus mutans Ingbritt.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Amino Acid Sequence↗
PubMed1998

Primary structure and molecular modeling of mistletoe lectin I from Viscum album.

The first three-dimensional structure of the ribosome inactivating protein mistletoe lectin I (ML-I) from Viscum album has been modeled on the basis of the X-ray structure of castor bean ricin from Ricinus communis. The relative high sequence homology and conserved secondary structure enabled accurate modeling. The 196 sequence changes between ML-I and ricin could be accomodated with only little pertubation in the main chain folding. A close comparison of the primary structures of ML-I and ricin is given and the effects of the sequence changes are elucidated on the basis of the modeled three-dimensional structure. Differences have been identified in the vicinity of the active site, in the high affinity galactose binding site and in the interface between the A and B chains, which might account for the reduced cytotoxicity of ML-I.

Amino Acid Sequence↗
PubMed1999

Molecular modeling of human procathepsin E: analysis of salt-bridge interactions between propeptide and enzyme segment.

A three-dimensional structural model of human cathepsin E zymogen (e. g., procathepsin E) has been constructed based upon the crystal structures of porcine pepsinogen. The overall protein folding features of the model are similar to those observed in the template structures. The propeptide packs into the active-site cleft with a similar secondary structural pattern and is associated with enzyme segment by salt-bridges, hydrogen bondings, and hydrophobic interactions. As judged from the model, the salt bridges present between the propeptide and enzyme segment show remarkable variations compared to porcine pepsinogen and human progastricin structures. Mapping of these interactions revealed that human procathepsin E might engage a different structural motif (alpha-helix;12P-19P) for protecting/blocking of catalytic site compared to pepsinogen and progastricin.

Amino Acid Sequence↗
PubMed2001

Molecular model for the binary complex of uropepsin and pepstatin.

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.

Binding Sites↗
PubMed2001

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII.

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.

Amino Acid Sequence↗
PubMed1997

Leishmania major: molecular modeling of cysteine proteases and prediction of new nonpeptide inhibitors.

The crystal structures of papain, cruzain, and human liver cathepsin B were used to build homology-based enzyme models of a cathepsin L-like cysteine protease (cpL) and a cathepsin B-like cysteine protease (cpB) from the protozoan parasite Leishmania major. Although structurally a member of the cathepsin B subfamily, the L. major cpB is not able to cleave synthetic substrates having an arginine in position P2. This biochemical property correlates with the prediction of a glycine instead of a glutamic acid at position 205 (papain numbering). The modeled active sites of the L. major cpB and cpL were used to screen the Available Chemicals Directory (a database of about 150,000 commercially available compounds) for potential cysteine protease inhibitors, using DOCK3.5. Based on both steric and force field considerations, 69 compounds were selected. Of these, 18 showed IC50's between 50 and 100 microM and 3 had IC50's below 50 microM. A secondary library of compounds, originally derived from a structural screen against the homologous protease of Plasmodium falciparum (falcipain), and subsequently expanded by combinatorial chemistry, was also screened. Three inhibitors were identified which were not only effective against the L. major protease but also inhibited parasite growth at 5-50 microM.

Animals↗
PubMed1993

Structure and molecular model refinement of pig pancreatic alpha-amylase at 2.1 A resolution.

The previously reported structural model of porcine pancreatic alpha-amylase has been corrected and improved by a genuinely independent structure solution. The electron density map was established by multiple isomorphous replacement (m.i.r.; using 5 derivatives) and subsequent solvent-flattening at 2.8 A resolution. The sequence was built into the well-defined regions of the m.i.r. map; this partial model was refined using a simulated annealing refinement method with phase restraints. Phase combination of m.i.r. phases and phases of the partial model allowed the completion of the model. The final refinement was based on 29,838 independent reflections in the 8 to 2.1 A resolution range. A final R-factor of 15.6% was obtained with a model obeying standard geometry within 0.014 A in bond lengths and 2.8 degrees in bond angles. The final model consists of all 496 amino acid residues, 1 calcium ion, 1 chloride ion and 353 water molecules. The model is described in detail; the calcium and chloride binding sites are characterized.

Animals↗
PubMed1997

Pentameric and decameric structures in solution of serum amyloid P component by X-ray and neutron scattering and molecular modelling analyses.

Human serum amyloid P component (SAP) is a normal plasma glycoprotein and the precursor of amyloid P component which is a universal constituent of the abnormal tissue deposits in amyloidosis. X-ray and neutron scattering data showed that pentameric or decameric ring structures for SAP in solution are readily distinguished. Further neutron data collection showed that SAP pentamers were reproducibly obtained in the presence of Ca2+ at pH 5.5 or in the presence of methyl 4,6-O-(1-carboxyethylidene)-beta-d-galactopyranoside (MObetaDG) and Ca2+ at pH 6.0 to 8.0, while SAP decamers were obtained in the presence of EDTA between pH 5.5 and 8.0. SAP pentamers have a mean X-ray RG of 3.99(+/-0.11) nm and a mean neutron RG of 3.69(+/-0.12) nm in 100% 2H2O. SAP decamers have a mean X-ray RG of 4.23(+/-0.12) nm and a mean neutron RG of 4.09(+/-0.14) nm in 100% 2H2O. The absorption coefficients of SAP pentamers and decamers differ by 10%. If we infer that the two alpha-helical A-faces are in contact with each other in the SAP decamer, the lack of structural change of the decamer with pH may be explained by the absence of His residues from the A-face of the SAP pentamer, and the change in absorption coefficients is compatible with the presence of Trp residues at this A-face. The rigid ring structure of pentameric SAP provided a test of scattering curves calculated from crystal structures. The only structural unknown is the orientation of the five chemically homogeneous oligosaccharide chains relative to the protein, but extended oligosaccharide structures were found to account for its scattering curve. X-ray scattering curves were best calculated using a hydrated structure, while neutron scattering curves were best calculated using an unhydrated structure. The outcome of these analyses was used to model the structure of decameric SAP. The evaluation of 640 structures for two SAP pentamers brought face-to-face to form SAP decamers gave better curve fits for structures in which the two A-faces were in contact with each other, in which it is likely that the two pentamers were out of alignment by a rotation of 36 degrees and the oligosaccharide chains were extended.

Carbohydrate Conformation↗
PubMed1999

Molecular modeling of the three-dimensional architecture of the RNA component of yeast RNase MRP.

RNase mitochondrial RNA processing (MRP) is a ribonucleoprotein endoribonuclease that is involved in RNA processing events in both the nucleus and the mitochondria. The MRP RNA is both structurally and evolutionarily related to RNase P, the ribonucleoprotein endoribonuclease that processes the 5'-end of tRNAs. Previous analysis of the RNase MRP RNA by phylogenetic analysis and chemical modification has revealed strikingly conserved secondary structural elements in all characterized RNase MRP RNAs. Utilizing successive constraint modeling and energy minimization I derived a three-dimensional model of the yeast RNase MRP RNA. The final model predicts several notable features. First, the enzyme appears to contain two separate structural domains, one that is highly conserved among all MRP and P RNAs and a second that is only conserved in MRP RNAs. Second, nearly all of the highly conserved nucleotides cluster in the first domain around a long-range interaction (LRI-I). This LRI-I is characterized by a ubiquitous uridine base, which points into a cleft between these two structural domains generating a potential active site for RNA cleavage. Third, helices III and IV (the yeast equivalent of the To-binding site) model as a long extended helix. This region is believed to be the binding site of shared proteins between RNase P and RNase MRP and would provide a necessary platform for binding these seven proteins. Indeed, several residues conserved between the yeast MRP and P RNAs cluster in the central region of these helixes. Lastly, characterized mutations in the MRP RNA localize in the model based on their severity. Those mutations with little or no effect on the activity of the enzyme localize to the periphery of the model, while the most severe mutations localize to the central portion of the molecule where they would be predicted to cause large structural defects. Press.

Bacillus subtilis↗
PubMed1999

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies.

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.

Alcohol Dehydrogenase↗
PubMed1999

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study.

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

Amino Acid Sequence↗
PubMed2000

Conformational changes during the assembly of factor B from its domains by (1)H NMR spectroscopy and molecular modelling: their relevance to the regulation of factor B activity.

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments in the presence of activated C3 (C3b or C3(H(2)O)). The Ba fragment contains three short consensus/complement repeat domains, while the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain, all three of which are implicated in multisite contacts with C3. The upfield-shifted signals in the (1)H NMR spectra of factor B, the Ba and Bb fragments, and the vWF-A and SP domains were used as sensitive conformational probes of their structures. Temperature studies and pH titrations showed that the Ba fragment and the vWF-A and SP domains had conformationally mobile structures. The comparison of the NMR spectra of the SP domains of both factor B and factor D showed that the factor D linewidths were broader than those for factor B, which may result from a range of proteolytically inactive conformations of factor D in the absence of substrate. The NMR spectra from the separate vWF-A and SP domains in combination with that of the Ba fragment generally accounted for that of intact factor B, apart from the perturbation of an upfield-shifted signal from the Ba fragment. A new upfield-shifted signal was observed in the Bb fragment that was not detected in the spectra for the vWF-A or SP domains or intact factor B. Ring current calculations based on homology models or crystal structures predicted that buried hydrophobic methyl-aromatic interactions probably accounted for the upfield-shifted signals, with many arising from the N-terminal subdomain of the SP domain to which the C terminus of the vWF-A domain is directly linked. It was concluded that: (1) the conformation of the free SP domain is better ordered in solution than that of factor D; (2) the conformation of the Ba fragment is affected by its incorporation into factor B; and (3) the proximity of the vWF-A and SP domains within the Bb fragment leads to a conformational change in which conserved charged residues may be important. Allosteric structural rearrangements in the SP domain as the result of its interactions with the vWF-A domain or the Ba fragment provide an explanation of the regulation of the catalytic activity of factor B.

Allosteric Regulation↗
PubMedDate not supplied

Molecular model of an actin filament capped by a severing protein.

Gelsolin is a six-domain protein with a wide array of actin regulating activities. Despite the growing body of structural data on this protein, little is known about how it binds F-actin during severing and capping. In this paper we have combined data from X-ray crystallography, NMR, and electron microscopy to develop a model of an actin filament capped by a severing protein. The protein which we have modeled is G1/alpha A1-2, a genetically engineered molecule containing domains from both gelsolin and alpha-actinin. In the capped filament, domains G1 and alpha A1-2 of the hybrid severing protein bind two adjacent monomers along the long-pitch F-actin helix. The distance spanning these domains suggests the need for a flexible linker between them. By analogy, this implies that the gelsolin deletion mutant G1-3 contacts the same two monomers in the capped filament and suggests that the linker between G1 and G2 plays a crucial role in severing and capping.

Actinin↗
PubMed2000

Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling.

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.

Bacterial Proteins↗