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Biomedical subjects

I A Wilson

Publications and source records attributed to I A Wilson.

At least 127 records · Page 7Linked to original sources
PubMed1992

Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.

Animals↗
PubMed1991

Structural aspects of antibodies and antibody-antigen complexes.

The structures of several Fab fragments and Fab-antigen complexes have now been solved at high resolution. These structures of antibodies in complex with proteins, peptides and various other haptens have enabled us to gain insights into the structural basis of immune recognition. Early structures of Fab fragments with and without bound haptens showed the antibody combining sites to be pockets or grooves. More recent Fab-protein complex structures have shown the antibody-antigen interactions to be more extensive with flatter, more undulating binding surfaces. We have solved the structures of three Fab fragments in their native form and as complexes with their respective antigens. Two of these are anti-peptide Fab fragments, the other an anti-progesterone Fab. Comparison of the free and bound structures indicates small but significant changes in the antibody on ligand binding. An analysis of the Fab complexes solved so far indicates that the antibodies can have very differently shaped binding sites, depending on the antigen.

Antibodies↗
PubMed1990

Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 A.

The three-dimensional structures of an antibody to a peptide and its complex with the peptide antigen have been determined at 2.8 A resolution. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with two molecules per asymmetric unit, whereas the complex crystals are hexagonal with one molecule per asymmetric unit. The Fab' and the Fab'-peptide complex structures have been solved independently by molecular replacement methods and have crystallographic R factors of 0.197 and 0.215, respectively, with no water molecules included. The amino-terminal portion of the peptide sequence (NH2-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron density map of the Fab'-peptide complex and adopts a well-defined type II beta-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is alpha-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding of the Fab' to conformationally altered forms of the native Mhr or to apo-Mhr. Immunological mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural analysis. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 A2 and 540 A2, respectively. These two structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were observed when peptide was bound, there were some small but significant side chain and main chain rearrangements.

Amino Acid Sequence↗
PubMed1989

Preliminary crystallographic data and primary sequence for anti-peptide Fab' B13I2 and its complex with the C-helix peptide from myohemerythrin.

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a = b = 142.5 A, c = 101.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and Z = 1. The native Fab' crystallizes in space group P212121 with unit cell dimensions a = 98.0 A, b = 151.7 A, c = 80.8 A, alpha = beta = gamma = 90 degrees, and Z = 2. Both crystal forms diffract to beyond 2.6 A resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary X-ray diffraction studies of a complex between interleukin-2 and a soluble form of the p55 component of the high affinity interleukin-2 receptor.

Human recombinant interleukin-2 (IL-2) and a soluble recombinant form of the human p55 (Tac antigen) component of the IL-2 receptor (IL-2R) have been cocrystallized in 1.7-1.8 M ammonium sulfate, in the pH range 7.0-8.2. Variously glycosylated forms of both receptor and ligand can be cocrystallized under those conditions. The best crystals of the putative receptor-ligand complex involve the enzymatically desialylated receptor and unglycosylated IL-2. These crystals belong to the trigonal space group P3(1)2(1) or its enantiomorph, with unit cell dimensions a = b = 91 A and c = 119 A, and diffract to 3.5 A resolution. There is one receptor-ligand complex asymmetric unit, with a Matthews coefficient of 2.7, assuming the presence of one IL-2 molecule-receptor molecule. Interestingly, in addition to IL-2 (Mr = 14,000), the p55 IL-2 receptor (Mr = 44,000) and two fragments of the receptor, of apparent Mr = 35,000 and 25,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crystals are enriched in a reducible dimeric form of the desialylated receptor (apparent Mr = 90,000), as compared with protein solution from which the crystals grow. The overall amino acid content in the crystals is consistent with a 1:1 ratio of receptor to ligand. A native data set has been collected on a multiwire area detector and the search for suitable heavy atom derivatives is in progress.

Crystallization↗
PubMed1989

Preliminary crystallographic investigations of glycinamide ribonucleotide transformylase.

Crystals of glycinamide ribonucleotide transformylase have been grown from 0.4 to 1 M ammonium sulfate, 0.6 to 1 M sodium-potassium phosphate, or 0.65 to 1 M citrate in the pH range 4.5-7.0. The single crystals display variable morphology with varying pH. The crystals belong to the orthorhombic space group C222 with cell dimensions a = 141.4 A, b = 98.2 A, c = 103.5 A. Co-crystals have also been obtained in the presence of the inhibitor 5,8-dideazafolate (KI = 18 microM) under similar crystallization conditions. Crystals of a chemically modified enzyme, iodinated at Cys-21, were grown under similar conditions within the pH range 6.5-7.0. These crystals are isomorphous with the unmodified enzyme. Crystals suitable for high resolution (less than 2.5 A) x-ray diffraction studies have been obtained for each of the above.

Acyltransferases↗
PubMed1989

Surgical management of adrenal disease.

This study reviews the surgical management of adrenal disease over a 23 year period in a Dublin teaching hospital. Nineteen patients were diagnosed as having either Medullary or Cortical disease. Ultrasound and CAT Scan proved highly accurate in pre-operative tumour localisation. This analysis highlights the atypical presentation of phaeochromocytoma, the accuracy of Ultrasound and CAT Scan in tumour localisation and the importance of peri-operative monitoring.

Adolescent↗
PubMed1988

Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin.

X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 A) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P2(1), with unit cell dimensions a = 90.3 A, b = 82.9 A, c = 73.4 A, beta = 122.5 degrees for the native Fab and a = 63.9 A, b = 73.0 A, c = 49.1 A, beta = 120.6 degrees for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.

Amino Acid Sequence↗
PubMed1988

Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

Adenylyl Cyclases↗
PubMed1987

Crystallographic analysis of the three-dimensional structure of baboon alpha-lactalbumin at low resolution. Homology with lysozyme.

The crystal structure of baboon alpha-lactalbumin has been determined at 6 A and at 4.5 A (0.6 nm and 0.45 nm) resolution by the method of isomorphous replacement. The principal derivative was prepared by reducing a disulphide bridge in the crystals and inserting a mercury atom. Detailed comparison of the electron-density maps with corresponding maps of hen egg-white lysozyme shows that they are closely similar, with correlation coefficients of 0.57 and 0.44 at 6 A and 4.5 A resolution respectively. This result, in accordance with earlier predictions based upon comparisons of amino-acid sequences, provides further evidence that class C lysozymes and alpha-lactalbumins are homologous proteins and it is in keeping with the hypothesis that the alpha-lactalbumins evolved from a lysozyme precursor.

Amino Acid Sequence↗
PubMed1987

Crystallization and preliminary crystallographic data for an antiprogesterone monoclonal antibody Fab' and steroid-Fab' complexes.

Crystals of an Fab' from an antiprogesterone monoclonal antibody (IgG1) have been grown from 1.5 M-ammonium sulfate, pH 5.5 to 8.5. The single crystals are hexagonal rods, space group P6222 (or P6422), with cell dimensions a = b = 135.2 A, and c = 124.2 A. Cocrystals of the Fab' with progesterone, pregnanedione and other related steroids have been grown. The complex crystals have different morphology but are isomorphous with the native crystals. A hydroxy-progesterone derivative obtained by substituting an iodo-benzoyl group at the 11 alpha-hydroxyl position looks promising as a suitable heavy-atom candidate in addition to other potential conventional heavy-atom derivatives. All crystals diffract to at least 2.8 A resolution and are suitable for high-resolution X-ray diffraction studies.

Animals↗
PubMed1987

Digital ischaemia and palpable pedal pulses.

Digital ischaemia in the presence of an otherwise well-perfused foot in the non-diabetic patient presents diagnostic problems especially as the manifestations are frequently evanescent. Eighty such patients (sixty men and twenty women aged 55-84 years) who presented to St. Laurence's Hospital from 1982 to 1985 inclusive were evaluated. Micro-embolization was inferred from the demonstration of a cardiac (20) or an arterial source (42) in 62 of 80 patients. In 14 of the remaining 18 patients a malignancy was diagnosed, which was found to be visceral cancer in 11 cases. Indium platelet scanning revealed platelet aggregation in 14 of 26 patients examined. Laboratory evidence of abnormalities of platelet aggregation or blood coagulation was found in only 6 patients. There have been no further symptoms in 37 patients who have been treated surgically or in 17 of 21 patients who have been treated with either anticoagulation or antiplatelet therapy.

Aged↗
PubMed1987

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin.

At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.

Chemical Precipitation↗
PubMed1987

Analysis of an anti-progesterone antibody: variable crystal morphology of the Fab' and steroid-Fab' complexes.

Anti-progesterone monoclonal antibodies are being used for structural studies of antibody-antigen interaction, for their ability to block pregnancy shortly after fertilization, and for hormone immunoassay. A mouse anti-progesterone monoclonal Fab' fragment has been crystallized in its native form and co-crystallized with seven different, but structurally related, steroids. The crystals show interesting preferences in their crystal morphology, depending on the bound steroid ligand. The X-ray crystallographic analysis of this Fab', complexed with a series of related steroid ligands, should reveal details of the chemistry of antibody-antigen union and provide insights into how steroids interact with proteins.

Animals↗
PubMed1986

Selection by site-directed antibodies of small regions of peptides which are ordered in water.

High resolution nuclear magnetic resonance has been used to study a short peptide which corresponds to the antigenic region of a larger peptide immunogen. This work shows that there is a strong conformational preference for a Type II beta-turn in solution. This observation has implications for the nature of the immunogenicity and antigenicity of peptide antigens as well as for the more general question of protein-folding mechanisms.

Antibodies, Monoclonal↗