Biomedical subjects

P T Wingfield

Publications and source records attributed to P T Wingfield.

At least 19 recordsLinked to original sources

PubMed2005

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope.

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.

Antibodies, Monoclonal↗

PubMed2004

Energetics of domain-domain interactions and entropy driven association of beta-crystallins.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.

Amino Acid Sequence↗

PubMed2004

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

Chromatography, Gel↗

PubMed2004

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-Crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

Chromatography, Gel↗

PubMed2003

Diversity of core antigen epitopes of hepatitis B virus.

Core antigen (cAg), the viral capsid, is one of the three major clinical antigens of hepatitis B virus. cAg has been described as presenting either one or two conformational epitopes involving the "immunodominant loop." We have investigated cAg antigenicity by cryo-electron microscopy at approximately 11-A resolution of capsids labeled with monoclonal Fabs, combined with molecular modeling, and describe here two conformational epitopes. Both Fabs bind to the dimeric external spikes, and each epitope has contributions from the loops on both subunits, explaining their discontinuous nature: however, their binding aspects and epitopes differ markedly. To date, four cAg epitopes have been characterized: all are distinct. Although only two regions of the capsid surface are accessible to antibodies, local clustering of the limited number of exposed peptide loops generates a potentially extensive set of discontinuous epitopes. This diversity has not been evident from competition experiments because of steric interference effects. These observations suggest an explanation for the distinction between cAg and e-antigen (an unassembled form of capsid protein) and an approach to immunodiagnosis, exploiting the diversity of cAg epitopes.

Cryoelectron Microscopy↗

PubMed2003

Characterization of a conformational epitope on hepatitis B virus core antigen and quasiequivalent variations in antibody binding.

We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to approximately 10-A resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides-two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid-T=3 and T=4 particles-present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from approximately 100 to approximately 0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.

Antibodies, Monoclonal↗

PubMed2001

Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-met interaction facilitates signaling.

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.

Animals↗

PubMed2001

An easy way to include weak alignment constraints into NMR structure calculations.

We have recently shown that an energy penalty for the incorporation of residual tensorial constraints into molecular structure calculations can be formulated without the explicit knowledge of the Saupe orientation tensor (Moltke and Grzesiek. J. Biomol. NMR, 1999, 15, 77-82). Here we report the implementation of such an algorithm into the program X-PLOR. The new algorithm is easy to use and has good convergence properties. The algorithm is used for the structure refinement of the HIV-1 Nef protein using 252 dipolar coupling restraints. The approach is compared to the conventional penalty function with explicit knowledge of the orientation tensor's amplitude and rhombicity. No significant differences are found with respect to speed, Ramachandran core quality or coordinate precision.

Algorithms↗

PubMed2001

Peptides corresponding to the heptad repeat motifs in the transmembrane protein (gp41) of human immunodeficiency virus type 1 elicit antibodies to receptor-activated conformations of the envelope glycoprotein.

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.

Amino Acid Sequence↗

PubMed2000

Monomer-dimer equilibrium of normal and modified beta A3-crystallins: experimental determination and molecular modeling.

Beta- and gamma-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the betagamma-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine betaA3-crystallin (rbetaA3) was compared to that of modified betaA3-crystallins with either an N-terminal deletion of residues 1 to 29 (rbetaA3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine gammaB-crystallin (rbetaA3cp). All three proteins exhibited reversible monomer-dimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium: 6.62 x 10(-6) M (rbetaA3), 0.86 x 10(-6) M (rbetaA3cp), and 1.83 x 10(-7) M (rbetaA3tr). Homology modeling of betaA3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions betaA3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.

Amino Acid Sequence↗

PubMed2000

HIV-1 rev depolymerizes microtubules to form stable bilayered rings.

We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44-49 nm in external diameter, 3.4-4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg(2+) and is inhibited by maytansine. The interaction involves the NH(2)-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH(2)-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection.

Amino Acid Sequence↗

PubMed2000

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia.

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.

AIDS Dementia Complex↗

PubMed2000

Molecular architecture of bacteriophage T4 capsid: vertex structure and bimodal binding of the stabilizing accessory protein, Soc.

T4 encodes two dispensable proteins that bind to the outer surface of the mature capsid. Soc (9 kDa) stabilizes the capsid against extremes of alkaline pH and temperature, but Hoc (40 kDa) has no perceptible effect. Both proteins have been developed as display platforms. Their positions on the hexagonal surface lattice of gp23*, the major capsid protein, were previously defined by two-dimensional image averaging of negatively stained electron micrographs of elongated variant capsids. We have extended these observations by reconstructing cryo-electron micrographs of isometric capsids produced by a point mutant in gene 23, for both Hoc+.Soc+ and Hoc+.Soc- phages. The expected T = 13 lattice was observed, with a single Hoc molecule at the center of each gp23* hexamer. The vertices are occupied by pentamers of gp24*: despite limited sequence similarity with gp23*, the respective monomers are similar in size and shape, suggesting they may have the same fold. However, gp24* binds neither Hoc nor Soc; in situ, Soc is visualized as trimers at the trigonal points of the gp23* lattice and as monomers at the sites closest to the vertices. In solution, Soc is a folded protein ( approximately 10% alpha-helix and 50-60% beta sheet) that is monomeric as determined by analytic ultracentrifugation. Thus its trimerization on the capsid surface is imposed by a template of three symmetry-related binding sites. The observed mode of Soc binding suggests that it stabilizes the capsid by a clamping mechanism and offers a possible explanation for the phenotype of osmotic shock resistance.

Bacteriophage T4↗

PubMed2000

HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase.

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

Amino Acid Sequence↗

PubMed2000

Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes.

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to deriveresidual dipolar couplings for human HIV-1 Nef and ubiquitin.

Acrylic Resins↗

PubMed2000

The structure of the transcriptional antiterminator NusB from Escherichia coli.

We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.

Amino Acid Sequence↗

PubMed2000

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

Amino Acid Sequence↗

PubMed1999

Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions.

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30's apparent inhibition of the HIV-1 integrase, MAP30's ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30's anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30's glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

Anti-HIV Agents↗