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P M Colman

Publications and source records attributed to P M Colman.

At least 19 recordsLinked to original sources
PubMed1998

Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors.

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

Acetamides↗
PubMed1997

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases.

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4 degrees C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18 degrees C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Binding Sites↗
PubMed1997

Virus versus antibody.

Variation in the proteins produced by animal viruses allows the virus to reinfect the same host, but is constrained by the requirement to maintain critical viral functions, in particular engagement with cellular receptors. The fundamental characteristics of proteins and their interactions with each other suggest that this may not be so much of a constraint at all.

Antibodies, Viral↗
PubMed1997

Electrostatic complementarity at protein/protein interfaces.

Calculation of the electrostatic potential of protein-protein complexes has led to the general assertion that protein-protein interfaces display "charge complementarity" and "electrostatic complementarity". In this study, quantitative measures for these two terms are developed and used to investigate protein-protein interfaces in a rigorous manner. Charge complementarity (CC) was defined using the correlation of charges on nearest neighbour atoms at the interface. All 12 protein-protein interfaces studied had insignificantly small CC values. Therefore, the term charge complementarity is not appropriate for the description of protein-protein interfaces when used in the sense measured by CC. Electrostatic complementarity (EC) was defined using the correlation of surface electrostatic potential at protein-protein interfaces. All twelve protein-protein interfaces studied had significant EC values, and thus the assertion that protein-protein association involves surfaces with complementary electrostatic potential was substantially confirmed. The term electrostatic complementarity can therefore be used to describe protein-protein interfaces when used in the sense measured by EC. Taken together, the results for CC and EC demonstrate the relevance of the long-range effects of charges, as described by the electrostatic potential at the binding interface. The EC value did not partition the complexes by type such as antigen-antibody and proteinase-inhibitor, as measures of the geometrical complementarity at protein-protein interfaces have done. The EC value was also not directly related to the number of salt bridges in the interface, and neutralisation of these salt bridges showed that other charges also contributed significantly to electrostatic complementarity and electrostatic interactions between the proteins. Electrostatic complementarity as defined by EC was extended to investigate the electrostatic similarity at the surface of influenza virus neuraminidase where the epitopes of two monoclonal antibodies, NC10 and NC41, overlap. Although NC10 and NC41 both have quite high values of EC for their interaction with neuraminidase, the similarity in electrostatic potential generated by the two on the overlapping region of the epitopes is insignificant. Thus, it is possible for two antibodies to recognise the electrostatic surface of a protein in dissimilar ways.

Antigen-Antibody Reactions↗
PubMed1996

Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme.

We previously isolated a variant of the influenza virus NWS/G70C, with a decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en in vitro, which has a mutation in one of the conserved residues of the neuraminidase Glu 119 to Gly. Despite the mutation, purified neuraminidase demonstrated the same specific activity as the parent neuraminidase. In contrast, characterization of a similar mutant by another group revealed a low specific activity of the enzyme. We confirm here that the specific activity of our variant is the same as that of the parent, but report that this mutation makes the enzyme inherently unstable, at high and low temperatures, either on the virion or as purified neuraminidase. Thus, for a valid determination of specific activity the concentration of native NA needs to be determined at the time of enzyme assay. Structurally, the instability may be partially explained by the introduction of a side chain (Gly), which carries a greater entropy penalty in condensation of the structure from the unfolded to the folded state and this, together with the loss of stabilizing interaction between Glu 119 and its neighbors in the active site, is not compensated for by the water molecule occupying the position of the carboxylate group (6).

Animals↗
PubMed1995

Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en.

A variant of the influenza virus NWS/G70C has been generated which has decreased sensitivity in vitro to the neuraminidase-specific inhibitor, 4-guanidino-Neu5Ac2en. The virus is 1000-fold less sensitive to the 4-guanidino-Neu5Ac2en in a plaque assay, but only 10-fold less sensitive to 4-amino-Neu5Ac2en. In an enzyme inhibition assay 250-fold more drug was needed to achieve inhibition comparable to that observed with the parent virus. In contrast to the plaque assay, the virus was fully sensitive to 4-amino-Neu5Ac2en in the enzyme inhibition assay. Kinetic analysis of 4-guanidino-Neu5Ac2en binding demonstrated that the variant no longer exhibited the slow binding characteristic seen with the parent and other influenza viruses and inhibition by Neu5Ac2en was also decreased. However, binding to 4-amino-Neu5Ac2en remained the same as the parent. Sequence analysis of this virus revealed a mutation at a previously conserved site in the enzyme active site of the neuraminidase, Glu 119 to Gly. Crystallographic analysis of the mutant neuraminidase with and without bound inhibitor confirmed this mutation and suggested that the reduced affinity for the 4-guanidino-Neu5Ac2en derives partly from the loss of a stabilizing interaction between the guanidino moiety and the carboxylate at residue 119, and partly from alterations to the solvent structure of the active site.

Animals↗
PubMed1995

Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.

Antiviral Agents↗
PubMed1994

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody.

BACKGROUND: While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS: The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS: The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.

Amino Acid Sequence↗
PubMed1994

Structure of phaseolin at 2.2 A resolution. Implications for a common vicilin/legumin structure and the genetic engineering of seed storage proteins.

The refinement to 2.2 A resolution of the three-dimensional structure of the seed storage protein phaseolin from the French bean (Phaseolus vulgaris) via an alternative crystal form is described. The refined structure reveals details of the molecule hitherto unobserved and in particular we identify the structural role of conserved residues within the broader 7 S (vicilin) family of seed storage proteins. On this basis we are able to postulate a canonical model for the structure of the 7 S proteins. This model in turn provides a means for interpreting the structure of the 11 S (legumin) family of seed storage proteins, for which no X-ray diffraction data are available. The 11 S proteins are shown to bear a much closer relationship to the 7 S proteins than was previously recognized. The canonical model of the 7 S protein structure also provides a basis for proposing engineered mutations of these proteins with the goal of enhancing nutritional and functional properties.

Amino Acid Sequence↗
PubMed1994

The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli.

BACKGROUND: N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives. RESULTS: The structure of the enzyme from Escherichia coli has been determined to 2.2 A resolution by X-ray crystallography. The enzyme is shown to be a tetramer, in which each subunit consists of an alpha/beta-barrel domain followed by a carboxy-terminal extension of three alpha-helices. CONCLUSIONS: The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase.

Aldehyde-Lyases↗
PubMed1994

Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex.

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4 degrees and 20 degrees C. At higher protein concentrations (approximately 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20 degrees C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains. A close interaction between two symmetry-related scFv suggests that they may have crystallized as dimers.

Animals↗
PubMed1994

Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities.

The three-dimensional structures of (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII and (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII from barley have been determined by x-ray crystallography at 2.2- to 2.3-A resolution. The two classes of polysaccharide endohydrolase differ in their substrate specificity and function. Thus, the (1-->3)-beta-glucanases, which are classified amongst the plant "pathogenesis-related proteins," can hydrolyze (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls and may therefore contribute to plant defense strategies, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall hydrolysis during mobilization of the endosperm in germinating grain or during the growth of vegetative tissues. Both enzymes are alpha/beta-barrel structures. The catalytic amino acid residues are located within deep grooves which extend across the enzymes and which probably bind the substrates. Because the polypeptide backbones of the two enzymes are structurally very similar, the differences in their substrate specificities, and hence their widely divergent functions, have been acquired primarily by amino acid substitutions within the groove.

Base Sequence↗
PubMed1994

Influenza virus neuraminidase: structure, antibodies, and inhibitors.

The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.

Antibodies↗
PubMed1994

Structure-based drug design.

There are now many successful examples of the design of new ligands based on knowledge of target protein structures. In most cases those ligands are unsuitable as drugs because of problems of toxicity, stability or bioavailability. The past twelve months have also seen the description of the structures of many proteins which are either known to be targets for existing drugs or have clear potential to be utilized in therapy.

Drug Design↗
PubMed1993

Shape complementarity at protein/protein interfaces.

A new statistic Sc, which has a number of advantages over other measures of packing, is used to examine the shape complementarity of protein/protein interfaces selected from the Brookhaven Protein Data Bank. It is shown using Sc that antibody/antigen interfaces as a whole exhibit poorer shape complementarity than is observed in other systems involving protein/protein interactions. This result can be understood in terms of the fundamentally different evolutionary history of particular antibody/antigen associations compared to other systems considered, and in terms of the differing chemical natures of the interfaces.

Antigen-Antibody Reactions↗
PubMed1993

Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.

The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.

Amino Acid Sequence↗
PubMed1993

Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.

A model is proposed for the three-dimensional structure of the paramyxovirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the enzyme-active center of influenza virus neuraminidase. The influenza virus enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.

Amino Acid Sequence↗