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P M Colman

Publications and source records attributed to P M Colman.

At least 55 records · Page 3Linked to original sources
PubMed1989

Three-dimensional structures of influenza virus neuraminidase-antibody complexes.

X-ray diffraction analysis of crystals of a monoclonal Fab fragment NC41 bound to a viral antigen, influenza virus neuraminidase, shows an epitope involving five surface loops of the antigen. In addition it reveals an unusual pairing pattern between the domains of light and heavy chains in the variable module of the antibody. We interpret this result to imply that association with antigen can induce changes in the quaternary structure of the Fab, through a sliding of domains at the variable light/variable heavy chains (VL-VH) interface. In addition, Fab binding has altered the conformation of some of the surface loops of the antigen. The structure of the NC10 Fab-neuraminidase complex has now also been solved. It binds an epitope that overlaps the NC41 epitope. In this structure, there is no electron density for the C-module of the Fab fragment, implying it is disordered in the crystal lattice. The implications of these, and other antibody-antigen structures, for immune recognition are discussed.

Antigen-Antibody Complex↗
PubMed1988

Structure of an escape mutant of glycoprotein N2 neuraminidase of influenza virus A/Tokyo/3/67 at 3 A.

The three-dimensional structure of the membrane glycoprotein neuraminidase of an escape mutant of the influenza virus strain A/Tokyo/3/67 has been determined to 3 A (1 A = 0.1 nm) resolution by X-ray diffraction. The mutant virus, selected by growing the virus in the presence of a monoclonal antibody to the neuraminidase, is shown to have undergone a single amino acid change of lysine to glutamic acid at residue 368. The three-dimensional structure of the neuraminidase is identical with that reported for A/Tokyo/3/67, except for a purely local adjustment of the structure at position 368.

Amino Acid Sequence↗
PubMed1987

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus.

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.

Amino Acid Sequence↗
PubMed1987

Distribution of sequence differences in influenza N9 neuraminidase of tern and whale viruses and crystallization of the whale neuraminidase complexed with antibodies.

Neuraminidase genes from A/tern/Australia/G70C/75 (H11N9) and A/whale/Maine/1/84 (H13N9) influenza viruses have been sequenced. Seventy-two nucleotide changes were found, 17 of which result in changes in the amino acid sequence of the neuraminidase; 3 in the stalk region and 14 in the heads. To our surprise, all of the sequence changes in the head region are located on the base of the neuraminidase tetramer, resulting in conservation of antigenic sites on top of the neuraminidase which vary extensively in human influenza virus neuraminidase. Whale N9 neuraminidase, like tern N9 neuraminidase, possesses high levels of hemagglutinating activity but, unlike the tern neuraminidase, failed to form large well-ordered crystals. However, when the neuraminidase was complexed with Fab fragments of monoclonal antibodies, which were made against the tern N9 neuraminidase, large crystals of the complexes were obtained which diffract X-rays to beyond 3 A.

Amino Acid Sequence↗
PubMed1987

Crystals of antibodies complexed with influenza virus neuraminidase show isosteric binding of antibody to wild-type and variant antigens.

We describe here, for the first time, crystals of antibodies bound to a viral antigen which diffract X-rays to beyond 3 A. Crystals have been grown of Fab fragments of monoclonal antibody NC41, complexed with influenza virus neuraminidase (NA) of the N9 subtype and with a variant of N9 NA having a sequence change of Asn to Asp at position 331. This reduces, but does not abolish, the binding of NC41 antibody (in the case of another variant, Ser 371 to Leu, binding of NC41 antibody appears to be abolished). We are presenting data on the three-dimensional structure of these two complexes which indicates that NC41 antibody binds isosterically to the wild type and variant neuraminidase molecules.

Antibodies, Monoclonal↗
PubMed1987

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.

Antibodies, Monoclonal↗
PubMed1986

Electron and X-ray diffraction studies of influenza neuraminidase complexed with monoclonal antibodies.

Complexes of influenza virus neuraminidase both with antigen-binding (Fab) fragments and with whole monoclonal antibody molecules have been crystallized. Uniformly thin platelet microcrystals suitable for structure analysis by electron diffraction, yielding reflections to approximately 4.3 A resolution, have been grown from one neuraminidase-Fab complex, that of N9 neuraminidase with 32/3 Fab, and thicker crystals of a second neuraminidase-Fab complex (N9 neuraminidase-NC35 Fab) diffract X-rays to approximately 4.0 A resolution. Electron microscope lattice images of microcrystals both of Fab and of immunoglobulin G complexed with neuraminidase have been interpreted in terms of negatively stained images of the respective individual complex protomers. The sites of binding of the antibodies to the antigen are consistent with the notion that single amino acid changes observed in monoclonal variants of neuraminidase occur in binding epitopes for the antibody used for their selection.

Antibodies, Monoclonal↗
PubMed1985

Gene and protein sequence of an influenza neuraminidase with hemagglutinin activity.

An influenza virus neuraminidase (NA) of the N9 subtype also has hemagglutinin (HA) activity (W. G. Laver, P. M. Colman, R. G. Webster, V. S. Hinshaw, and G. M. Air (1984), Virology 137, 314-323). To determine sequence relationships between this NA and other known NA and HA subtype sequences, and as a necessary step toward a complete structure determination, we have cloned a full-length copy of the coding sequence of the N9 NA of influenza virus A/tern/Australia/G70C/75 into the plasmid pUC9 using SalI linkers. The gene was sequenced by directed subcloning into the single-stranded phage vectors M13mp19 and M13mp18 and use of the dideoxy procedure. Most of the NA sequence was also obtained by direct protein sequencing of tryptic peptides. The N9 NA has 43 and 44% homology when compared to N1 or N2 sequences, respectively. There is no significant homology to any known HA sequence, or to the HN protein of the paramyxovirus SV5. Like the other NA molecules, the N9 NA is anchored in the membrane by an N-terminal hydrophobic region, from which biologically active heads can be released by pronase.

Amino Acid Sequence↗
PubMed1984

Influenza virus neuraminidase with hemagglutinin activity.

Isolated intact influenza virus neuraminidase (NA) molecules of the N9 subtype have been found to possess hemagglutinin (HA) activity which, at equivalent protein concentration, was fourfold higher than that of isolated hemagglutinin molecules of the H3 subtype. The amino-terminal sequence of the N9 NA is the same as in neuraminidases of the eight other influenza A virus NA subtypes previously reported. Viruses possessing N9 NA therefore have two different HA activities and antibody to either HA or NA alone was incapable of inhibiting hemagglutination by the virus. However, antibody to the HA of an H1N9 virus neutralized its infectivity as effectively as it neutralized H1N1 or H1N2 viruses whose neuraminidases have no HA activity. (Antibodies to N9 NA did not neutralize the infectivity of viruses with N9 neuraminidase). 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid inhibited N9 NA activity but had no effect on the HA activity of the isolated N9 NA. One interpretation of this result would be that the HA and NA activities are located in separate sites. Pronase-released N9 NA heads form crystals suitable for X-ray diffraction studies and preliminary data to 2.9 A establish the space group as cubic, I432 with cell dimension a = 184 A. Data extend to beyond 1.9 A resolution, and these will be collected in the future.

Animals↗
PubMed1983

The disulphide bonds of an Asian influenza virus neuraminidase.

The arrangement of the disulphide bonds in the pronase-released neuraminidase heads of the Asian influenza virus A/Tokyo/3/67 have been examined by cyanogen bromide fragmentation, enzymic digestion and diagonal peptide mapping. There are 9 intrachain disulphide bridges and one interchain bridge which links pairs of monomers at the distal end of the stalk region of the neuraminidase tetramer. The disulphide bond arrangements of the remaining 3 half-cystine residues in the membrane-embedded stalk region of the neuraminidase were not examined.

Amino Acid Sequence↗
PubMed1983

Crystallization of phaseolin from Phaseolus vulgaris.

Three different types of crystals were grown from phaseolin, the major storage body protein from French bean. Type I crystals are cubes with space group symmetry P432, a = 67 A. Type II crystals are bipyramids with a rounded basal plane and belong to space group P2(1)2(1)2, a = 128 A, b = 136 A, and c = 162 A. Type III crystals are rhombs grown from phytic acid-free protein. The space group symmetry is P2(1)2(1)2(1), a = 113 A, b = 136 A, and c = 89 A. Both Type II and III crystals are suitable for high resolution x-ray study.

Crystallization↗
PubMed1983

Three-dimensional structure of the Mcg IgG1 immunoglobulin.

The three-dimensional structure of an IgG1(lambda) immunoglobulin from a patient (Mcg) with amyloidosis was determined at 6.5-A resolution with X-ray diffraction techniques. The protein crystallized from water in the space group C2221, with a = 87.8, b = 111.3 and c = 186.3 A; the crystallographic asymmetric unit was a half-molecule consisting of one light and one heavy chain. The structure was solved by the multiple isomorphous replacement method with five heavy-atom derivatives. Electron density maps were interpreted with the aid of a protein modeling system used in conjunction with an Evans and Sutherland Picture System II graphics station. IgG1 molecules were tightly packed in the crystal lattice, with numerous intermolecular contacts. The two-fold axis relating identical halves of each molecule was found to be parallel to the y crystallographic axis. Electron density modules collectively representing one molecule were identified as three lobes representing the two antigen-binding (Fab) arms and the Fc region. An interchain disulfide bond connecting the two CL domains was located on the molecular diad and used as a landmark in the interpretation of the electron density map. A computer graphics method was developed to produce a solid image model of the IgG1 molecule in any prescribed orientation.

Computers↗
PubMed1980

The structure of cucurbitin: subunit symmetry and organization in situ.

The low-resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X-ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point-group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry. Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry.

Crystallization↗