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R L Brady

Publications and source records attributed to R L Brady.

At least 37 records · Page 2Linked to original sources
PubMed1999

One sequence, four folds: transitions between an ensemble of metastable folds for the N-terminal domain of CD2.

Recombinant forms of the N-terminal domain of the cell adhesion receptor CD2 adopt a variety of olds by exchange of beta-sheets between adjacent polypeptide chains. Although these interdigitated forms are normally metastable, we have used site-directed mutagenesis to alter the kinetics of formation and relative stabilities of these states, leading to spontaneous formation of monomeric, dimeric, trimeric and tetrameric intertwined folded states. A characteristic feature of these fold-disorder-alternative fold transitions is the independence of each domain folding event, as deduced from kinetic analysis of folding data. Structures for fully interdigitated trimeric and tetrameric forms have been modelled, consistent with both the crystallographic and kinetic data. Although the biological role of these alternative folded states remains unclear, these structures form a remarkable demonstration of the fluidity of structure generated from a single polypeptide chain.

Amino Acid Sequence↗
PubMed1998

Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction.

BACKGROUND: Proteins belonging to the phosphatidylethanolamine-binding protein (PEBP) family are highly conserved throughout nature and have no significant sequence homology with other proteins of known structure or function. A variety of biological roles have previously been described for members of this family, including lipid binding, roles as odorant effector molecules or opioids, interaction with the cell-signalling machinery, regulation of flowering plant stem architecture, and a function as a precursor protein of a bioactive brain neuropeptide. To date, no experimentally derived structural information has been available for this protein family. In this study we have used X-ray crystallography to determine the three-dimensional structure of human PEBP (hPEBP), in an attempt to clarify the biological role of this unique protein family. RESULTS: The crystal structures of two forms of hPEBP have been determined: one in the native state (at 2.05 A resolution) and one in complex with cacodylate (at 1.75 A resolution). The crystal structures reveal that hPEBP adopts a novel protein topology, dominated by the presence of a large central beta sheet, and is expected to represent the archaetypal fold for this family of proteins. Two potential functional sites have been identified from the structure: a putative ligand-binding site and a coupled cleavage site. hPEBP forms a dimer in the crystal with a distinctive dipole moment that may orient the oligomer for membrane binding. CONCLUSIONS: The crystal structure of hPEBP suggests that the ligand-binding site could accommodate the phosphate head groups of membrane lipids, therefore allowing the protein to adhere to the inner leaf of bilipid membranes where it would be ideally positioned to relay signals from the membrane to the cytoplasm. The structure also suggests that ligand binding may lead to coordinated release of the N-terminal region of the protein to form the hippocampal neurostimulatory peptide, which is known to be active in the development of the hippocampus. These studies are consistent with a primary biological role for hPEBP as a transducer of signals from the interior membrane surface.

Amino Acid Sequence↗
PubMed1998

Engineering an intertwined form of CD2 for stability and assembly.

The amino-terminal domain of CD2 has the remarkable ability to fold in two ways: either as a monomer or as an intertwined, metastable dimer. Here we show that it is possible to differentially stabilize either fold by engineering the CD2 sequence, mimicking random mutagenesis events that could occur during molecular evolution. Crystal structures of a hinge-deletion mutant, which is stable as an intertwined dimer, reveal domain rotations that enable the protein to further assemble to a tetramer. These results demonstrate that a variety of folds can be adopted by a single polypeptide sequence, and provide guidance for the design of proteins capable of further assembly.

Animals↗
PubMed1997

VL:VH domain rotations in engineered antibodies: crystal structures of the Fab fragments from two murine antitumor antibodies and their engineered human constructs.

The crystal structures of two pairs of Fab fragments have been determined. The pairs comprise both a murine and an engineered human form, each derived from the antitumor antibodies A5B7 and CTM01. Although antigen specificity is maintained within the pairs, antigen affinity varies. A comparison of the hypervariable loops for each pair of antibodies shows their structure has been well maintained in grafting, supporting the canonical loop model. Detailed structural analysis of the binding sites and domain arrangements for these antibodies suggests the differences in antigen affinity observed are likely to be due to inherent flexibility of the hypervariable loops and movements at the VL:VH domain interface. The four structures provide the first opportunity to study in detail the effects of protein engineering on specific antibodies.

Animals↗
PubMed1996

Structure of the Fab fragment of a monoclonal antibody specific for carcinoembryonic antigen.

The three-dimensional structure of the Fab fragment of the murine monoclonal antibody A5B7, which is specific for carcinoembryonic antigen (CEA) a protein expressed on carcinoma cell surfaces, has been determined. The structure was solved by molecular replacement and has been refined to an R factor for 21.2% (all data 8-2.1 A). The conformation of the hypervariable loops, which form the antigen binding site, are consistent with canonical loop predictions. Hypervariable loop H3 is unusual in surface exposing many hydrophobic groups at the expense of burying an aspartic acid in the protein core. Other regions of the structure that influence the conformation of the binding site are identified. This structure forms a basis for analysing the effects of amino-acid substitutions in both hypervariable and framework regions in engineering studies of the A5B7 antibody.

Journal Article↗
PubMed1996

Antigenicity of truncated forms of the human immunodeficiency virus type 1 envelope glycoprotein.

Chinese hamster ovary (CHO) cell lines secreting a series of truncated forms of human immunodeficiency virus type 1 (HIV-1) IIIB (clone BH10) gp12O were assembled. Using purified glycoproteins, we demonstrated the functional and structural integrity of these truncates by their reactivity with both sCD4 and anti-gp 120 monoclonal antibodies (MAbs). Deletion of the Vl, V2 and V3 regions had minimal effects on CD4 binding, but deletion of the NH2 terminus affected the folding of the truncated molecule. Deletion of either V1/V2 or V1/V2/V3 regions led to enhanced recognition by some, but not all, MAbs mapping to the CD4 binding site. In contrast, deletion of the V1/V2 regions had no effect on the ability of V3-specific MAbs to bind to the truncate. These results support the use of truncated forms of gp12O as components of potential HIV vaccines.

Animals↗
PubMed1995

One sequence, two folds: a metastable structure of CD2.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.0-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.

Animals↗
PubMed1994

Crystal structure of an extracellular fragment of the rat CD4 receptor containing domains 3 and 4.

BACKGROUND: CD4 is a transmembrane protein on the surface of T lymphocytes that interacts with MHC class II proteins at the surface of accessory cells, and is involved in the triggering of the lymphocytes by foreign antigens. It is also the major receptor for the human immunodeficiency virus. The extracellular portion of CD4 was predicted to contain four immunoglobulin superfamily domains and this has been confirmed by X-ray crystallography, but no detailed structure of domains 3 and 4 has been available. RESULTS: We now report the expression of a form of rat CD4 containing only domains 3 and 4, its crystallization, and the refinement and analysis of its structure by X-ray crystallography with 2.6 A spacing data. Both domains show variations in core residues when compared with immunoglobulin domains. Features of the structure are discussed with respect to the structure of the complete extracellular part of CD4 and its function. CONCLUSIONS: Domains 3 and 4 of CD4 show considerable similarity to domains 1 and 2, although there is a 25 degrees rotation in the relative positions of the domains with respect to one another. The absence of the disulphide bond in domain 3 is associated with an alteration in the packing of the beta-sheets, which may be important for interactions with domain 2 in the overall receptor structure. The location of N-linked glycosylation on one face of domain 3 appears to preclude the dimerization that is observed in antibodies.

Amino Acid Sequence↗
PubMed1993

CD4 and the immunoglobulin superfamily.

The CD4 membrane glycoprotein was one of the first cell surface antigens to be identified using monoclonal antibodies. It was shown to have a central role in the control of the recognition of foreign proteins by T lymphocytes and later as a receptor for the human immunodeficiency virus (HIV). The analysis of the amino acid sequence of CD4 showed that the extracellular region comprised four regions with sequence similarities to immunoglobulin domains. The structure of domains 3 and 4 of CD4 has been determined by X-ray crystallography and, like domains 1 and 2 previously determined, these have typical immunoglobulin-like folds. The results are discussed with respect to the identification of other domains with immunoglobulin-like folds from amino acid sequence data, and the evolution of CD4.

Amino Acid Sequence↗
PubMed1993

Crystal structure of domains 3 and 4 of rat CD4: relation to the NH2-terminal domains.

The CD4 antigen is a membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigens and is also a receptor for the human immunodeficiency virus. the extracellular portion of CD4 is predicted to fold into four immunoglobulin-like domains. The crystal structure of the third and fourth domains of rat CD4 was solved at 2.8 angstrom resolution and shows that both domains have immunoglobulin folds. Domain 3, however, lacks the disulfide between the beta sheets; this results in an expansion of the domain. There is a difference of 30 degrees in the orientation between domains 3 and 4 when compared with domains 1 and 2. The two CD4 fragment structures provide a basis from which models of the overall receptor can be proposed. These models suggest an extended structure comprising two rigid portions joined by a short and possibly flexible linker region.

Amino Acid Sequence↗
PubMed1992

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.

Amino Acid Sequence↗
PubMed1991

Solution of the structure of Aspergillus niger acid alpha-amylase by combined molecular replacement and multiple isomorphous replacement methods.

The crystal structure of Aspergillus niger acid alpha-amylase was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The atomic coordinates of Aspergillus oryzae (TAKA) alpha-amylase (entry 2TAA in the Protein Data Bank) and experimental diffraction data from a new monoclinic crystal form of TAKA alpha-amylase, were used during the procedure. Sequence identity between the two proteins is approximately 80%. The atomic parameters derived from the molecular replacement solution were too inaccurate to initiate least-squares crystallographic refinement. The molecular model was extensively revised against the experimental electron density map calculated at 3 A resolution. Subsequent crystallographic refinement of this model using synchrotron data to 2.1 A resolution led to a conventional R factor of 16.8%. The structure conforms well to expected stereochemistry with bond lengths deviating from target values by 0.031 A, and planar groups showing a root-mean-square deviation from ideal planes of 0.025 A.

Aspergillus niger↗
PubMed1991

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells.

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.

Animals↗
PubMed1990

Crystallization of a soluble form of the rat T-cell surface glycoprotein CD4 complexed with Fab from the W3/25 monoclonal antibody.

The structure of the T-lymphocyte cell surface glycoprotein CD4 is of considerable biological and medical interest. Recombinant rat CD4 expressed in soluble form in mammalian cells and complexed with W3/25 monoclonal Fab fragments formed crystals that diffract to 3.5 A and have the orthorhombic space group P2(1)2(1)2 or P2(1)2(1)2(1). The unit cell has dimensions a = 317 A, b = 161 A and c = 41.8 A and the asymmetric unit consists of two CD4:Fab complexes. These crystals are of suitable quality for X-ray diffraction analysis.

Animals↗
PubMed1990

Expression of HIV-1 gp120 and human soluble CD4 by recombinant baculoviruses and their interaction in vitro.

The soluble domains of the envelope glycoprotein of HIV-1 (gp120) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 10(9) cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gp120-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

Animals↗