Biomedical subjects

P T Wingfield

Publications and source records attributed to P T Wingfield.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1999

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.

Animals↗

PubMed1999

Proteolytic processing of HIV-1 protease precursor, kinetics and mechanism.

Previously it was demonstrated using a model precursor that processing at the N terminus of the HIV-1 protease (PR) precedes processing at its C terminus. We now show the expression, purification, and kinetics of the autoprocessing reaction of a PR precursor linked to 53 amino acids of the native flanking transframe region (DeltaTFP-p6(pol)) of Gag-Pol and containing its two native cleavage sites. The PR contains the two cysteine residues exchanged to alanines, mutations that do not alter the kinetics or the structural stability of the mature PR. DeltaTFP-p6(pol)-PR, which encompasses the known PR inhibitor sequence Glu-Asp-Leu within DeltaTFP, undergoes cleavage at the DeltaTFP/p6(pol) and p6(pol)/PR sites in two consecutive steps to produce the mature PR. Both DeltaTFP-p6(pol)-PR and p6(pol)-PR exhibit low intrinsic enzymatic activity. The appearance of the mature PR is accompanied by a large increase in catalytic activity. It follows first-order kinetics in protein concentration with a rate constant of 0.13 +/- 0.01 min(-1) in 0.1 M acetate at pH 4.8. The pH-rate profile for the observed first-order rate constant is bell-shaped with two ionizable groups of pK(a) 4.9 and 5.1. The rate constant also exhibits approximately 7-fold higher sensitivity to urea denaturation as compared with that of the mature PR, suggesting that the cleavage at the N terminus of the PR domain from the precursor leads to the stabilization of the dimeric structure.

Amino Acid Sequence↗

PubMed1999

Biophysical and functional characterization of full-length, recombinant human tissue inhibitor of metalloproteinases-2 (TIMP-2) produced in Escherichia coli. Comparison of wild type and amino-terminal alanine appended variant with implications for the mechanism of TIMP functions.

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.

Escherichia coli↗

PubMed1999

The crystal structure of the SIV gp41 ectodomain at 1.47 A resolution.

Cell membrane fusion by human (HIV) and simian (SIV) immunodeficiency viruses is mediated by the envelope glycoproteins gp120 and gp41. Although the precise mechanism of the fusion process is unknown, the ectodomain of gp41 is thought to undergo dramatic rearrangement from its prefusogenic state. To elucidate this process further, the crystal structure of the SIV gp41 ectodomain (residues 27-149) was determined at 1.47 A resolution and is reported herein. It is the most accurate and complete structure of a retroviral gp41 ectodomain determined to date. The rod-like trimeric structure of SIV gp41 comprises three parallel N-terminal alpha-helices assembled as a coiled coil in the center with three antiparallel C-terminal alpha-helices packed on the outside connected by highly flexible loops. Portions of the loops in all three monomers are crystallographically disordered and could not be accurately modeled. The core of the structure is similar (but not identical) to those of smaller HIV/SIV gp41 segments previously determined by X-ray crystallography with root mean square deviations in main chain atoms of less than 1.0 A. The crystal structure differs more substantially from the reported NMR solution structure of the identical SIV construct. The mechanisms of viral fusion and the inhibition by peptides are discussed in the context of the three-dimensional structure.

Amino Acid Sequence↗

PubMed1999

Separation and crystallization of T = 3 and T = 4 icosahedral complexes of the hepatitis B virus core protein.

The icosahedral nucleocapsid of human hepatitis B virus is a homopolymer of the dimeric capsid protein also known as hepatitis B core antigen or HBcAg. Purified capsid protein obtained from an Escherichia coli expression system was reassembled into a mixture of T = 3 and T = 4 icosahedral particles consisting of 90 and 120 dimers, respectively. The two types of capsid were separated on a preparative scale by centrifugation through a sucrose gradient. In addition to this heterogeneity, the capsid protein has three cysteines, one of which has a great propensity for forming disulfide bonds between the two subunits, forming a dimer. To eliminate heterogeneity arising from oxidation, alanines were substituted for the cysteines. T = 3 and T = 4 capsids crystallized under similar conditions. Crystals of T = 3 capsids diffracted to approximately 8 A resolution; crystals of T = 4 capsids diffracted to 4 A resolution.

Capsid↗

PubMed1999

Is human thioredoxin monomeric or dimeric?

We have examined the molecular weight and rotational correlation time of human thioredoxin by analytical ultracentrifugation and NMR spectroscopy, respectively. Two variants of human thioredoxin were studied, namely human thioredoxin identical in amino acid sequence to the one whose NMR structure we previously determined (C62A, C69A, C73A, M74T) and human thioredoxin (C62A, C69A, C73A, M74) containing the wild-type amino acid methionine at position 74. In both cases, the experimental data indicate that the predominant species is monomeric and we find no evidence for the existence of a well-defined dimeric form as was observed in the recently reported crystal structure (Weichsel et al., 1996) of human thioredoxin and the C73S mutant.

Humans↗

PubMed1998

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy.

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 A, enabling direct visualization of alpha-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i. e., insertion of peptides, approximately 10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 A resolution. Hepatitis B virus capsids are icosahedral particles, approximately 300 A in diameter, made up of T-shaped dimers (subunit Mr, 16-21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and -7 (in the residual propeptide) in the "e-antigen" form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Amino Acid Sequence↗

PubMed1998

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.

Amino Acid Sequence↗