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V R Harley

Publications and source records attributed to V R Harley.

33 records · Page 2Linked to original sources
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

N9 neuraminidase complexes with antibodies NC41 and NC10: empirical free energy calculations capture specificity trends observed with mutant binding data.

X-ray crystallographic coordinates of influenza virus N9 neuraminidase complexed with monoclonal antibodies NC41 and NC10 [Tulip et al. (1992) J. Mol. Biol. 227, 122-148] served as a starting point for calculations aimed at estimating free energy changes (delta G) of complex formation between the two antibodies and the neuraminidase. Using an empirical function incorporating hydrophobic, electrostatic, and conformational entropy effects, we estimated contributions individual neuraminidase residues make to complex formation (delta G(residue)) and compared the calculated values to experimentally measured differences in antibody binding between the wild-type and mutated neuraminidases [Nuss et al. (1993) Proteins 15, 121-132; calculations done without prior knowledge of the experimental data]. A good correspondence was found between the calculated delta G(residue) values and the mutant binding data in that side chains with large calculated delta G contributions (delta G(residue) < -1 kcal/mol) lie at sites of mutation which cause a marked reduction in antibody binding, and side chains for which delta G(residue) > -1 kcal/mol are sites at which a mutation does not have a marked effect on binding. Because most of the delta G(residue) < -1 kcal/mol side chains also make hydrogen bonds/salt bridges with the antibody, the correspondence of the effect of antibody binding with these electrostatic interactions (18 out of 27 for NC41 and, tentatively, 5 out of 7 for NC10) is about as good as that with predicted energetic residues. All the delta G(residue) < -1 kcal/mol neuraminidase side chains cluster around the most protruding surface regions and are thus spread over different epitope segments. Surprisingly, different residues were found to make the most critical contributions to the NC41 and NC10 complex stabilities despite the fact that the NC41 and NC10 antigenic epitopes overlap, having approximately 70% of surface residues in common. It is thus possible, for two different antibodies, to recognize the same protein surface in strikingly different ways. As only a fraction of the neuraminidase residues appear to make large contributions to antibody binding, the results also support the hypothesis of a "functional" epitope in antigen-antibody interactions. Positive trends between both backbone rigidity and residue accessibility in the complexed state, and contributions of these residues to binding, were also observed for the NC41 complex.

Antibodies, Monoclonal↗
PubMed1994

The biochemical role of SRY in sex determination.

The human sex-determining gene on the Y chromosome, termed SRY, has recently been isolated by positional cloning; compelling evidence now exists equating SRY with the testis-determining factor, TDF. The SRY gene product is an HMG box protein whose DNA-binding activity is vital for testis formation as sex-reversed patients with SRY mutations lack this activity in vitro. The in vivo DNA target for SRY, however, remains elusive. Here, we show, by gel retardation analysis, that SRY recognises specific DNA sequences and that such sequences exist upstream of the AMH promoter, a potential downstream target for SRY. We also describe the DNA bending and cruciform DNA-binding functions of SRY and propose a model for the potential action of SRY in the "HMG-1-rich" mammalian nucleus.

Amino Acid Sequence↗
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↗
PubMed1992

High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal.

We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase).

Animals↗
PubMed1992

DNA binding activity of recombinant SRY from normal males and XY females.

The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.

Amino Acid Sequence↗
PubMed1992

SRY, like HMG1, recognizes sharp angles in DNA.

HMG boxes are DNA binding domains present in chromatin proteins, general transcription factors for nucleolar and mitochondrial RNA polymerases, and gene- and tissue-specific transcriptional regulators. The HMG boxes of HMG1, an abundant component of chromatin, interact specifically with four-way junctions, DNA structures that are cross-shaped and contain angles of approximately 60 and 120 degrees between their arms. We show here also that the HMG box of SRY, the protein that determines the expression of male-specific genes in humans, recognizes four-way junction DNAs irrespective of their sequence. In addition, when SRY binds to linear duplex DNA containing its specific target AACAAAG, it produces a sharp bend. Therefore, the interaction between HMG boxes and DNA appears to be predominantly structure-specific. The production of the recognition of a kink in DNA can serve several distinct functions, such as the repair of DNA lesions, the folding of DNA segments with bound transcriptional factors into productive complexes or the wrapping of DNA in chromatin.

Amino Acid Sequence↗
PubMed1992

A familial mutation in the testis-determining gene SRY shared by both sexes.

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRYF109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic background or environmental factors.

Amino Acid Sequence↗
PubMed1990

Characterisation of an avian influenza virus nucleoprotein expressed in E. coli and in insect cells.

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.

Animals↗
PubMed1990

Vaccinia virus expression and sequence of an avian influenza nucleoprotein gene: potential use in diagnosis.

The nucleoprotein (NP) gene from avian influenza strain A/Shearwater/Aust/1/72 (H6N5) was cloned, sequenced, and expressed in vaccinia virus for the production of potent sera in immunised rabbits. The NP gene is 1565 bp and shares greater than 95% amino acid sequence identity with other NPs of the avian subtype. The recombinant NP expressed by vaccinia virus comigrated with endogenous A/Shearwater/Aust/1/72 NP by Western blot analysis. Polyclonal rabbit sera raised against recombinant NP was evaluated in an antigen capture ELISA system as a potential diagnostic tool for the detection of avian influenza. All type A strains, comprising several HA and NA subtypes, but not type B nor other avian viruses, were detected.

Amino Acid Sequence↗
PubMed1990

Marfan syndrome: absence of type I or III collagen structural defects in 25 patients.

The structure and metabolism of type I and III collagens were studied in fibroblast cultures and dermis from 25 unrelated patients including 23 with typical Marfan syndrome and two infants with a very severe clinical form of this syndrome. Electrophoretic analysis of collagen alpha-chains, as well as one- and two-dimensional electrophoresis of collagen cyanogen bromide peptides, failed to show any evidence of primary structure defects or overmodification of lysine residues in these collagens. The proportion of hydroxylated prolyl residues in isolated alpha 1(I) chains was also normal. There was a minimal increase in the proportion of type III collagen produced by nine cultures. The findings in this study indicate that the underlying molecular defects in the patients studied are unlikely to involve the structure of the main fibrillar type I and III collagens.

Cells, Cultured↗
PubMed1989

Molecular cloning and analysis of the N5 neuraminidase subtype from an avian influenza virus.

The neuraminidase (NA) gene from the prototype N5 influenza virus, A/Shearwater/Australia/72, has been cloned and completely sequenced. An open reading frame of 1404 bp (468 amino acids) is flanked by 20-bp 5'- and 31-bp 3'-untranslated regions. The deduced amino acid sequence of the N5 gene was compared with sequences from N2, N1, N7, N8, and N9 subtypes. One hundred thirteen amino acid residues (24%) are completely conserved across subtypes and include active site residues, cysteines, potential glycosylation sites, and certain glycines which suggests that these subtypes share a common ancestor and adopt the same 3-D conformation. Three groups can be assigned from amino acid homologies: (i) N5, N8, N1; (ii) N7, N9; and (iii) N2 where the percentage identity within groups is 55-68% and between groups is 40-46%, the N5-N8 pair bearing the closest identity (68%). Phylogenetic analysis suggests that these groups diverged concurrently.

Amino Acid Sequence↗
PubMed1988

Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product.

The smaller dsRNA segment of the genome of infectious bursal disease virus (IBDV) encodes a single polypeptide of approximately 90 kDa (VP1). The consensus nucleotide sequence, derived from independent and overlapping cDNA clones, contains a single open reading frame which begins with an exact Kozak sequence and could encode a polypeptide of 878 amino acid residues. It has been suggested that VP1 could be the viral RNA-dependent RNA polymerase. A comparison of the predicted amino acid sequence of this protein with those of other DNA-dependent and ssRNA-dependent RNA polymerases has failed to reveal any homology between VP1 and the conserved regions in these enzymes. It is possible that the polypeptide encoded by the IBDV virus may represent a new class of polymerases which are involved in the replication of double-stranded RNA genomes.

Amino Acid Sequence↗