PubMed Health⌕ Search

Biomedical subjects

C J March

Publications and source records attributed to C J March.

At least 19 recordsLinked to original sources
PubMed1995

Direct evidence of a heterotrimeric complex of human interleukin-4 with its receptors.

The mode of binding of interleukin-4 (IL-4) to its two known receptors, specific receptor IL-4R and a shared receptor gamma c, was investigated using gel filtration and gel electrophoresis. A ternary complex between IL-4 and the soluble domains of the two receptors was shown to exist in solution. The association constant between gamma c and the stable complex of IL-4/sIL-4R is in the millimolar range, making the ternary complex a feasible target for crystallization studies.

Amino Acid Sequence↗
PubMed1995

Modeling protein loops using a phi i + 1, psi i dimer database.

We present an automated method for modeling backbones of protein loops. The method samples a database of phi i + 1 and psi i angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles phi i + 1 and psi i completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed phi i + 1, psi i pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N-terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of phi i + 1, psi i for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three-dimensional structures. Typically, 10,000-50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50-80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near-native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near-native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop-closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near-native loop conformations.

Amino Acid Sequence↗
PubMed1994

Homology modeling of divergent proteins.

A method is presented for homology modeling of proteins bearing weak sequence identity to proteins of known tertiary structure. To accommodate non-identical amino acids in the core region, the backbone of the structurally conserved core of the model protein is allowed to deviate from that of the template protein. We have expanded FOLDER, a distance geometry-based homology modeling method, to allow for such displacements in the structurally conserved core. Models are built by rigidly constraining the interatomic distances within a structurally conserved segment and by allowing the interatomic distances between these segments to vary by a "divergence factor". We test this method by simulating models of the beta-barrel domain D1 of CD4 and a four-helix bundle protein cytochrome b562 using the crystal structures of Bence-Jones protein and cytochrome c' as templates, respectively. In both cases, previously published structure-based sequence alignments were used for simulating models. The root-mean-square (r.m.s.) deviation of the backbone atoms in the common core between the templates and models was found to be a function of the imposed divergence factor. Our results demonstrate that this r.m.s. deviation results from the relative displacements of structurally conserved segments to accommodate the amino acid replacements in the core of the model protein. To test the integrity of the simulated structures we compared them with their respective crystal structures. The r.m.s. deviation of the backbone atoms in the core regions of the simulated models and their respective crystal structures is approximately 1.4 A. The r.m.s. deviation for all the backbone atoms in the models, including those in the structurally variable regions, which are modeled de novo, is 2.4 A for CD4 and 3.2 A for cytochrome b562 when compared with their respective X-ray structures.

Animals↗
PubMed1994

Stoichiometry of the complex of human interleukin-4 with its receptor.

A large number of cytokines have been shown to possess a four-helix bundle structure with a unique up-up-down-down connectivity. The receptors for this family of cytokines have been shown to be homologous as well, each possessing two tandem repeats of a fibronectin type III-like domain. The crystal structure of human growth hormone bound to the soluble portion of its receptor has served as the only experimentally-determined example of the interaction between the four-helix bundle cytokines and their receptors: two identical receptor subunits bind to different epitopes on the same growth hormone ligand. We have conducted a series of experiments to determine if this structural paradigm is true for interleukin-4 and interleukin-4 receptor. Native polyacrylamide gel electrophoresis and gel filtration chromatography reveal that interleukin-4 forms a tight 1:1 complex with the system.

Growth Hormone↗
PubMed1993

The high-resolution, three-dimensional solution structure of human interleukin-4 determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of approximately 15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three- and four-dimensional heteronuclear NMR spectroscopy. The structure is based on a total of 2973 experimental NMR restraints, comprising 2515 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.44 +/- 0.03 A for the backbone atoms, 0.83 +/- 0.03 A for all atoms, and 0.51 +/- 0.04 A for all atoms excluding disordered side chains. The N- and C-terminal residues (1-7 and 130-133, respectively) appear to be disordered. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The latter include a mini anti-parallel beta-sheet. A best fit superposition of the NMR structure of IL-4 with the 2.25 A resolution crystal structure [Wlodawer, A., Pavlovsky, A., & Gutschina, A. (1992) FEBS Lett. 309, 59-64] yields a backbone atomic rms difference of 1.37 A which can be mainly attributed to tighter packing of the helices in the crystal structure. This is indicated by an approximately 20% reduction in the axial separation of three pairs of helices (alpha A-alpha C, alpha A-alpha D, and alpha C-alpha D) in the crystal structure relative to the NMR structure and may reflect the greater flexibility of the molecule in solution which is reduced in the crystal due to intermolecular contacts. Comparison of the NMR structure of IL-4 with the X-ray structures of two other related proteins, granulocyte-macrophage colony stimulating factor [Diedrichs, K., Boone, T., & Karplus, P. A. (1992) Science 254, 1779-1782] and human growth hormone [de Vos, A. M., Ultsch, M., & Kossiakoff, A. A. (1992) Science 255, 306-312], that bind to the same hematopoietic superfamily of cell surface receptors reveals a remarkably similar topological fold, despite the absence of any significant overall sequence identity, and substantial differences in the relative lengths of the helices, the lengths and the nature of the various connecting elements, and the pattern and number of disulfide bridges.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.

We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.

Amino Acid Sequence↗
PubMed1992

Three-dimensional solution structure of human interleukin-4 by multidimensional heteronuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of recombinant human interleukin-4, a protein of 133 residues and 15.4 kilodaltons that plays a key role in the immune and inflammatory systems, has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The overall topology is remarkably similar to that of growth hormone and granulocyte-macrophage colony stimulating factor, despite the absence of any sequence homology, and substantial differences in the relative lengths of the helices, the length and nature of the various connecting elements, and the pattern of disulfide bridges. These three proteins, however, bind to cell surface receptors belonging to the same hematopoietic superfamily, which suggests that interleukin-4 may interact with its receptor in an analogous manner to that observed in the crystal structure of the growth hormone-extracellular receptor complex.

Amino Acid Sequence↗
PubMed1992

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

Amino Acid Sequence↗
PubMed1992

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure of human recombinant interleukin-4 (IL-4) has been investigated by three-dimensional (3D) 15N- and 13C-edited nuclear Overhauser (NOE) spectroscopy on the basis of the 1H, 15N, and 13C assignments presented in the preceding paper [Powers, R., Garrett, D. S., March, C. J., Frieden, E. A., Gronenborn, A. M., & Clore, G. M. (1992) Biochemistry (preceding paper in this issue)]. Based on the NOE data involving the NH, C alpha H, and C beta H protons, as well as 3JHN alpha coupling constant, amide exchange, and 13C alpha and 13C beta secondary chemical shift data, it is shown that IL-4 consists of four long helices (residues 9-21, 45-64, 74-96, and 113-129), two small helical turns (residues 27-29 and 67-70), and a mini antiparallel beta-sheet (residues 32-34 and 110-112). In addition, the topological arrangement of the helices and the global fold could be readily deduced from a number of long-range interhelical NOEs identified in the 3D 13C-edited NOE spectrum in combination with the spatial restrictions imposed by three disulfide bridges. These data indicate that the helices of interleukin-4 are arranged in a left-handed four-helix bundle with two overhand connections.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of the interleukin-1 beta converting enzyme.

Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary DNA encoding a protease that carries out this cleavage has been cloned. Recombinant expression in COS-7 cells enabled the cells to process precursor IL-1 beta to the mature form. Sequence analysis indicated that the enzyme itself may undergo proteolytic processing. The gene encoding the protease was mapped to chromosomal band 11q23, a site frequently involved in rearrangement in human cancers.

Amino Acid Sequence↗
PubMed1992

Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.

Amino Acid Sequence↗
PubMed1992

An approach to computer-aided inhibitor design: application to cathepsin L.

We have developed an approach to search for molecules that can be used as lead compounds in designing an inhibitor for a given proteolytic enzyme when the 3D structure of a homologous protein is known. This approach is based on taking the cast of the binding pocket of the protease and comparing its dimensions with that of the dimensions of small molecules. Herein the 3D structure of papain is used to model cathepsin L using the comparative modeling technique. The cast of the binding pocket is computed using the crystal structure of papain because the structures of papain and the model of cathepsin L are found to be similar at the binding site. The dimensions of the cast of the binding site of papain are used to screen for molecules from the Cambridge Structural Database (CSD) of small molecules. Twenty molecules out of the 80,000 small molecules in the CSD are found to have dimensions that are accommodated by the papain binding pocket. Visual comparison of the shapes of the cast and the 20 screened molecules resulted in identifying brevotoxin b, a toxin isolated from the 'red tide' dinoflagellate Ptycho brevis (previously classified as Gymonodium breve), as the structure that best fits the binding pocket of papain. We tested the proteolytic activity of papain and cathepsin L in the presence of brevotoxin b and found inhibition of papain and cathepsin L with Kis of 25 microM and 0.6 microM, respectively. We also compare our method with a more elaborate method in the literature, by presenting our results on the computer search for inhibitors of the HIV-1 protease.

Binding Sites↗
PubMed1991

Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.

Amino Acid Sequence↗
PubMed1991

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4.

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.

Amino Acid Sequence↗
PubMed1990

Expression cloning of a human granulocyte colony-stimulating factor receptor: a structural mosaic of hematopoietin receptor, immunoglobulin, and fibronectin domains.

We report the isolation from a placental library, of two cDNAs that can encode high affinity receptors for granulocyte colony-stimulating factor (G-CSF) when expressed in COS-7 cells. The cDNAs are predicted to encode integral membrane proteins of 759 and 812 amino acids in length. The predicted extracellular and membrane spanning sequences of the two clones are identical, as are the first 96 amino acids of their respective cytoplasmic regions. Different COOH termini of 34 or 87 residues are predicted for the two cDNAs, due apparently to alternate splicing. The receptor with the longer cytoplasmic domain is the closest human homologue of the murine G-CSF receptor recently described by Fukunaga et al. (Fukunaga, R., E. Ishizaka-Ikeda, Y. Seto, and S. Nagata. 1990. Cell. 61:341). A hybridization probe derived from the placental G-CSF receptor cDNA detects a approximately 3-kb transcript in RNAs isolated from placenta and a number of lymphoid and myeloid cells. The extracellular region of the G-CSF receptors is composed of four distinct types of structural domains, previously recognized in other cell surface proteins. In addition to the two domains of the HP receptor family-defining region (Patthy, L. 1990. Cell. 61:13) it incorporates one NH2-terminal Ig-like domain, and three additional repeats of fibronectin type III-like domains. The presence of both an NH2-terminal Ig-like domain and multiple membrane-proximal FN3-like domains suggests that the G-CSF receptor may be derived from an ancestral NCAM-like molecule and that the G-CSF receptor may function in some adhesion or recognition events at the cell surface in addition to the binding of G-CSF.

Amino Acid Sequence↗
PubMed1990

Expression cloning of a human Fc receptor for IgA.

IgA, the predominant isotype in secretions, mediates the neutralization and removal of environmental antigens from mucosal sites. Although cell surface receptors for the Fc region of IgA (Fc alpha R) have been implicated in a variety of immune effector mechanisms, the molecular features of Fc alpha R remain only marginally characterized. In this report, we describe the isolation of a clone from a myeloid cell line cDNA library that directs the expression of a cell surface molecule with IgA binding specificity. The cDNA encodes a peptide of Mr 30,000 including a putative transmembrane region with features atypical of conventional membrane-anchored proteins. Databank searches indicate that the human myeloid cell Fc alpha R sequence is unique, is a member of the immunoglobulin gene superfamily, and is related to Fc receptors for IgG (Fc gamma RI, II, and III) and IgE (Fc epsilon RI).

Amino Acid Sequence↗