Biomedical subjects

I D Campbell

Publications and source records attributed to I D Campbell.

At least 19 recordsLinked to original sources

PubMed1992

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

Amino Acid Sequence↗

PubMed1992

The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions.

The solution structure of the tenth type III module of fibronectin has been determined using nuclear magnetic resonance techniques. The molecule has a fold similar to that of immunoglobulin domains, with seven beta strands forming two antiparallel beta sheets, which pack against each other. Both beta sheets contribute conserved hydrophobic residues to a compact core. The topology is more similar to that of domain 2 of CD4, PapD, and the extracellular domain of the human growth hormone receptor than to that of immunoglobulin C domains. The module contains an Arg-Gly-Asp sequence known to be involved in cell adhesion. This tripeptide is solvent exposed and lies on a conformationally mobile loop between strands F and G, consistent with its cell adhesion function.

Amino Acid Sequence↗

PubMed1992

Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha.

The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.

Amino Acid Sequence↗

PubMed1992

Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments.

Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein. Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined. The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet. This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.

Amino Acid Sequence↗

PubMed1992

Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase.

Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis.

Animals↗

PubMed1992

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.

Amino Acid Sequence↗

PubMed1992

Solution structure of the fibrin binding finger domain of tissue-type plasminogen activator determined by 1H nuclear magnetic resonance.

The amino acid sequence of the first domain of tissue-type plasminogen activator (t-PA) includes eight residues that are highly conserved in the type 1 finger domains found in human fibronectin. A construct comprising 50 residues from this finger domain of t-PA has been expressed and its solution structure has been determined by two-dimensional nuclear magnetic resonance spectroscopy. A total of 782 experimental restraints consisting of 723 interproton distances derived from nuclear Overhauser effect measurements, 43 torsion angles, and 16 hydrogen bond restraints were used as the input for dynamical simulated annealing structure calculations. Twenty-eight structures were obtained that satisfied the experimental data with no single distance violation greater than 0.3 A. The average atomic root-mean-square distribution for the backbone atoms of the final structures was 0.41 (+/- 0.13) A for the well defined part of the structure (residues 4 to 47). The overall fold of the t-PA finger domain shows a striking similarity to that of the seventh type 1 repeat of human fibronectin with the side-chains of conserved residues lying in similar conformations. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the principal beta-sheet region in the t-PA finger domain. It is suggested that one face of this region may interact with parts of the complete t-PA protein.

Amino Acid Sequence↗

PubMed1992

Solution structure of the fifth repeat of factor H: a second example of the complement control protein module.

Modules which share the same consensus sequence are assumed to have common structural features, at the secondary and tertiary level. In order to test the extent of such similarities, it is necessary to examine the structures of several examples from each module family. Recently, the first three-dimensional structure of a complement control protein (CCP) module (the 16th repeat of human factor H, H16) was determined using a combination of two-dimensional NMR and simulated annealing [Norman, D.G., Barlow, P.N., Baron, M., Day, A.J., Sim, R.B., & Campbell, I.D. (1991) J. Mol. Biol. 219, 717-725]. Using the same techniques, the three-dimensional structure of a second CCP module (the 5th repeat of human factor H, H5) has now been determined. The primary sequence of H5 contains 17 residues which are identical and in equivalent position to those in H16. Thirteen of these 17 are part of the consensus sequence. The similarities between the secondary structure of H5 and that of H16 are extensive. This implies that the consensus sequence dictates a particular secondary structure. The tertiary structure of H5, a compact hydrophobic core wrapped in beta-strand and sheet, bears much overall resemblance to that of H16. However, there is a deletion in the first strand of H5, and an insertion in a loop, resulting in slightly shorter overall length. This is associated with a rearrangement of residues within the hydrophobic core. The side chain of the highly conserved Tyr29, which occupies a central position within the core of H16, lies on the periphery of the core of H5.

Amino Acid Sequence↗

PubMed1992

1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin.

The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.

Amino Acid Sequence↗

PubMed1992

Proliferation indices of invasive breast carcinomas after in vivo 5-bromo-2'-deoxyuridine labelling: a flow cytometric study of 75 tumours.

In vivo labelling of human breast tumours with 5-bromo-2'-deoxyuridine and analysis of proliferation by multiparameter flow cytometry is a new tool by which the labelling index, S phase duration (TS) and potential doubling time (Tpot) of the tumour can be estimated. Tumour specimens from 75 patients with invasive breast carcinoma were studied. Six tumours could not be analysed, and the TS and Tpot values could not be calculated for a further 18. The median labelling index (n = 69) was 4.2 per cent. The median TS (n = 51) was 8.7 h and the median Tpot (n = 51) was 8.2 days. The median intra-assay coefficient of variation for determination of the labelling index was 0.29 in five tumours. There were no significant differences in the total labelling index, TS or Tpot when patients were stratified according to lymph node status, tumour size, tumour grade or menopausal status.

Adult↗

PubMed1992

Three dimensional structure of the transmembrane region of the proto-oncogenic and oncogenic forms of the neu protein.

The neu proto-oncogene may be converted into a dominantly transforming oncogene by a single point mutation. Substitution of a valine residue at position 664 in the transmembrane region with glutamic acid activates the tyrosine kinase of the molecule and is associated with increased receptor dimerization. Previously we have proposed a model in which the glutamic acid side chain stabilizes receptor dimerization by hydrogen bonding. Other models have been proposed in which the mutation leads to a conformational change in the transmembrane region mimicking that assumed to occur following binding of a natural ligand. Synthetic peptides representing part of the transmembrane region were prepared. Some residues were replaced with serine in order to improve peptide solubility to allow purification and analysis. Both the peptides containing valine and glutamic acid dissolved in water and in an artificial lipid monolayer. The structures of the peptides were determined by NMR spectroscopy to be alpha-helical. No significant difference in conformation was observed between the two peptides. This result does not support the model proposing a conformational change. The receptor structures determined experimentally do allow alternative models involving receptor transmembrane region packing.

Amino Acid Sequence↗

PubMed1992

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.

Amino Acid Sequence↗

PubMed1992

Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.

Amino Acid Sequence↗

PubMed1991

Structure of domain 1 of rat T lymphocyte CD2 antigen.

The CD2 antigen is largely restricted to cells of the T-lymphocyte lineage and has been established as an important adhesion molecule in interactions between human T lymphocytes and accessory cells. In the adhesion reaction, CD2 on T cells binds to LFA-3 on other cells, with binding through domain 1 of CD2. CD2 can also be a target for the delivery of mitogenic signals to T lymphocytes cultured with combinations of anti-CD2 antibodies. Two predictions that are contradictory have been made for the structure of CD2 domain 1. One suggests an immunoglobulin (Ig) fold, on the basis of sequence patterns conserved in the Ig-superfamily (IgSF), whilst the other proposes a pattern of alternating alpha-helices and beta-strands, on the basis of secondary structure predictions. Thus CD2 domain 1 is an important test case for the validity of IgSF assignments based on sequence patterns. We report here the expression of domain 1 of rat CD2 in an Escherichia coli expression system and have determined a low-resolution solution structure by NMR spectroscopy.

Amino Acid Sequence↗

PubMed1991

Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR.

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Amino Acid Sequence↗

PubMed1991

The solution structure of human transforming growth factor alpha.

The solution structure of transforming growth factor alpha has been determined by a combination of high-resolution 1H-nuclear magnetic resonance and distance geometry and restrained molecular dynamics. The 382 restraints derived from the NMR experiments were used to calculate many distance geometry structures, which were then refined by restrained molecular mechanics. Five of these structures were further refined using a variety of methods. Comparison of independently measured parameters, such as calculated hydrogen bonding patterns and experimental amide exchange rates, have been used to evaluate the accuracy of the structures. Also, possible mechanisms to explain the pH-dependent conformational interconversion observed are suggested. Finally comparisons between this work and others on this topic have been made.

Amino Acid Sequence↗

PubMed1991

Solution structure of human insulin-like growth factor 1: a nuclear magnetic resonance and restrained molecular dynamics study.

The solution structure of human insulin-like growth factor 1 has been investigated with a combination of nuclear magnetic resonance and restrained molecular dynamics methods. The results show that the solution structure is similar to that of insulin, but minor differences exist. The regions homologous to insulin are well-defined, while the remainder of the molecule exhibits greater disorder. The resultant structures have been used to visualize the sites for interaction with a number of physiologically important proteins.

Amino Acid Sequence↗