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J Bajorath

Publications and source records attributed to J Bajorath.

At least 55 records · Page 3Linked to original sources
PubMed1997

Protein superfamily members as targets for computer modeling: the carbohydrate recognition domain of a macrophage lectin.

Members of protein superfamilies display similar folds, but share only limited sequence identity, often 25% or less. Thus, it is not straightforward to apply standard homology modeling methods to construct reliable three-dimensional models of such proteins. A three-dimensional model of the carbohydrate recognition domain of the rat macrophage lectin, a member of the calcium-dependent (C-type) lectin superfamily, has been generated to illustrate how information provided by comparison of X-ray structures and sequence-structure alignments can aid in comparative modeling when primary sequence similarities are low.

Amino Acid Sequence↗
PubMed1996

Mutational analysis of the CD6 binding site in activated leukocyte cell adhesion molecule.

The interaction between CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) mediates adhesion of thymocytes to thymic epithelial cells. The extracellular region of ALCAM includes five Ig-like domains, and its N-terminal V-like domain specifically binds to the membraneproximal scavenger receptor cysteine-rich domain of CD6. Previously, six ALCAM residues were identified by alanine scanning mutagenesis to contribute to the interaction with CD6. All of these residues mapped to the predicted A'GFCC'C" face of ALCAM's N-terminal domain. Here we describe the results of experiments designed to further study the CD6 binding site. Other mutagenesis experiments at four previously studied sites were carried out to better understand their importance for the interaction with CD6, and different receptor binding assays were employed to compare the contribution of these and other ALCAM residues to the CD6-ligand interaction. A total of ten new ALCAM mutants were prepared, and three additional residues were identified as critical for CD6 binding. These studies have enabled us to classify ALCAM residues according to their importance for binding and to describe the CD6 binding site in some detail.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

Comparison of CH1 domains in different classes of murine antibodies.

The CH1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG CH1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG CH1 domain structures, the IgA CH1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG CH1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for CH1 domains has been revised to be consistent with the three-dimensional alignments.

Amino Acid Sequence↗
PubMed1996

Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions.

T lymphocyte receptors CD28 and CTLA-4 bind costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. While distinct functional roles have been ascribed to each of these molecules, little is known about how they interact. To better characterize these interactions, we have used surface plasmon resonance to perform equilibrium and kinetic binding analyses of extracellular fragments of CD28/CTLA-4/CD80/CD86. We show that CTLA-4 and CD28 binding are both characterized by rapid kinetic on-rates and rapid dissociation rates. Native disulfide-linked homodimers of CD28 and CTLA-4 bound with two kinetically distinct binding sites, one of high avidity and slow dissociation and one of low avidity and more rapid dissociation. Monomeric CTLA-4 bound only with low affinity and rapid dissociation. Therefore, covalent dimerization of CTLA-4 is required for its high avidity binding. Oligomerization of CD80/CD86 is also required for high avidity CTLA-4 binding since CTLA-4 bound with low avidity to monomeric CD86. This contrasts with the ability of CD80/CD86 on antigen-presenting cells to bind CTLA4Ig with high avidity and predicts their organization as oligomers or clusters that permit multivalent binding. Thus, covalent receptor dimerization and ligand oligomerization are two key features of the CD28/CTLA-4/CD80/CD86 receptor system that control ligand binding and may regulate signal transduction by controlling the duration of receptor occupancy.

Abatacept↗
PubMed1996

Recognition of diverse proteins by members of the immunoglobulin superfamily: delineation of the receptor binding site in the human CD6 ligand ALCAM.

The CD6-ALCAM (activated leukocyte cell adhesion molecule) interaction, which mediates thymocyte--thymic epithelial cell adhesion, is a previously unobserved type of protein--protein interaction that involves members of the scavenger receptor cysteine rich protein superfamily (SRCRSF) and the immunoglobulin superfamily (IgSF). Targeted mutagenesis of ALCAM reveals that residues which constitute the CD6 binding site cluster on the predicted A'GFCC'C" face of its N-terminal Ig domain. These results, in conjunction with recent analyses of interactions involving other IgSF members, suggest that this region in IgSF cell surface proteins is most suitable to mediate interactions with different ligands irrespective of their structure. The CD6 binding site in ALCAM is conserved across species, and nonconserved residues in ALCAM and its murine homolog map to the beta-sheet face opposite to the CD6 binding site. This provides a molecular rationale for the inability to obtain murine monoclonal antibodies against the receptor binding domain which block the CD6-ALCAM interaction.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

A combinatorial library strategy for the rapid humanization of anticarcinoma BR96 Fab.

We have used a combinatorial mutagenesis strategy to humanize BR96, a monoclonal antibody that binds to the Lewis Y class of tumor antigens. This approach allows simultaneous assessment of hundreds of humanized variable regions to identify the molecules that best preserve affinity, thus overcoming the major drawback of current humanization procedures, the requirement to construct and analyze each humanized antibody separately. Murine residues of BR96 were mutated to human if they were solvent-exposed residues that did not participate in the formation of the antigen binding site and were not at the interface of the light and heavy chain. At positions that might be involved in binding to antigen, the choice between the murine and human residue was more difficult. Murine and human alternatives were incorporated into a combinatorial library at positions representing buried residues that might affect the structural integrity of the antigen binding site. By encoding this library of humanized BR96 Fabs in an M13 phage vector, we rapidly identified several candidates with nearly identical antigen binding, within 2-fold, of the chimeric Fab. Additional mutagenesis directed at sites suggested in the literature as potentially important for antigen binding in a similar anti-Lewis Y antibody yielded no further improvements.

Amino Acid Sequence↗
PubMed1996

The amino-terminal immunoglobulin-like domain of activated leukocyte cell adhesion molecule binds specifically to the membrane-proximal scavenger receptor cysteine-rich domain of CD6 with a 1:1 stoichiometry.

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

X-ray structure of the uncomplexed anti-tumor antibody BR96 and comparison with its antigen-bound form.

The X-ray structure of the uncomplexed human chimeric Fab' of the anti-tumor antibody BR96 has been determined at 2.6 A resolution. The structure has been compared with Lewis Y antigen-complexed structures of BR96 which were determined previously. The comparison reveals segmental motions and/or conformational rearrangements of three CDR loops (L1, L3, and H2), whereas CDR H3 does not undergo changes upon complexation despite its significant main-chain contacts to the carbohydrate antigen. In light of the uncomplexed chimeric Fab' structure reported here, the previously observed high mobility of the CL:CH1 domains of the complexed chimeric BR96 Fab is rationalized as a "swinging" motion approximately about the axis of the elbow bend.

Animals↗
PubMed1996

Modifying the specificity and activity of the Enterobacter cloacae P99 beta-lactamase by mutagenesis within an M13 phage vector.

A library of Enterobacter cloacae P99 beta-lactamase mutants was produced to investigate the importance of residues 286-290 for substrate binding and catalysis and to characterize mutants with altered specificities and activities for various 3'-substituted cephalosporins. This region of the enzyme is a component of the active site that has not been implicated as participating in the catalytic mechanism but, based on molecular modeling, should contact the 3' substituents of cephalosporins. Random mutagenesis was carried out within an M13 phage vector by hybridization mutagenesis, and the phage library could be highly enriched for active beta-lactamase genes by incubation of infected bacteria with beta-lactam antibiotics. The mutants were characterized by Michaelis-Menten kinetic analyses with several cephalosporin substrates and spanned a 25-fold range of k(cat), 24-fold range of K(m), and 6-fold range of k(cat)/K(m) values. All five amino acid positions were found to be permissive to substitution, but the active mutant proteins carried substitutions that likely maintained the structure of the region. Serine 287 was the least permissive to change, requiring small, uncharged residues for retention of catalytic activity. The variation of Michaelis-Menten kinetic parameters observed in these enzymes was shown to be significant in the context of in vitro cytotoxicity assays with the cephalosporin-doxorubicin prodrug C-Dox and is suitable for experiments to probe the relationship between enzyme kinetics and efficacy in enzyme-prodrug approaches to targeted therapy.

Bacteriophage M13↗
PubMed1996

Comparison of an antibody model with an X-ray structure: the variable fragment of BR96.

A model of the BR96 antibody variable regions is compared to two X-ray structures of a BR96-carbohydrate complex, independently determined after the model was built and analyzed. The comparison illustrates the opportunities and limitations of antibody modeling. Encouraging results were obtained for the prediction of single CDR loop conformations and for the outline of the BR96 antigen binding site. The comparison of CDR loop conformations in the two X-ray structures provides a realistic reference frame for the CDR loop predictions. CDR loop prediction accuracy is lower when not only conformational, but also positional criteria are taken into account.

Animals↗
PubMed1996

Structure-based modeling of the ligand binding domain of the human cell surface receptor CD23 and comparison of two independently derived molecular models.

CD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C-type lectin domain: immunoglobulin E (IgE), CD21, and the beta 2-integrins CD11b and CD11c. CD23 specifically interacts in a calcium-dependent manner, "lectin-like" with carbohydrate moieties expressed on CD21 and CD11b/c, but also "lectin-unlike" with protein epitopes on IgE. As a first step in analyzing the multiple binding specificities associated with CD23 in more detail, we report a detailed molecular model of the lectin-like domain of human CD23 (hCD23). The model was built based on information provided by X-ray structures of mannose binding protein (MBP) and E-selectin, both of which are members of the calcium-dependent (C-type) lectin superfamily. Sequence-structure comparisons suggest that hCD23 is structurally more similar to MBP than to E-selectin. The hCD23 model is compared to an independently derived model. Although the CD23-carbohydrate and CD23-protein interactions are both calcium dependent, analysis of the model suggests the presence of distinct binding sites for these ligands.

Amino Acid Sequence↗
PubMed1996

Classification of mutations in the human CD40 ligand, gp39, that are associated with X-linked hyper IgM syndrome.

The interaction between the T cell activation antigen gp39 and CD40, its receptor CD40 on B cells, plays a critical role in the regulation of humoral immune responses. Using a detailed three-dimensional model of the gp39 extracellular region, we have analyzed 20 mutations in gp39 that were, with one exception, isolated from patients with X-linked hyper IgM (XHIM) syndrome. On the basis of this analysis, the mutations were classified according to their predicted locations and effects. Twelve mutations are thought to compromise the gp39 structure by affecting interactions in hydrophobic core regions or at monomer interfaces, whereas seven others map closely to gp39 residues important for interaction with CD40. The latter mutations may thus, directly or indirectly, interfere with CD40 binding. One naturally occurring mutant whose carrier displays normal immune responses maps to a solvent-exposed position in a loop region of the molecule.

CD40 Antigens↗
PubMed1996

A molecular model of the carbohydrate recognition domain of a rat macrophage lectin and analysis of its binding site.

A three-dimensional model of the carbohydrate recognition domain of a rat macrophage C-type lectin has been constructed by comparative modeling and assessed by inverse folding analysis. Comparative modeling in the presence of low sequence similarity was based on information provided by comparison of X-ray structures and sequence-structure alignments. The sequence-structure compatibility of the model was sound. Its binding site was analyzed in comparison to the X-ray structure of a galactose-specific mutant of the mannose-binding protein. The specificity of the macrophage lectin was discussed in light of mutagenesis data on asialoglycoprotein receptors.

Animals↗
PubMed1996

Immune regulation by CD40 and its ligand GP39.

Over the past three years, CD40 and its ligand (gp39, CD40L, TBAM) have been shown to be essential for humoral immune responses to thymus-dependent antigens. However, as the tissue distribution widens for those cells that express CD40 and gp39, we can now show that this ligand-receptor pair also plays an important role in the selection of self-reactive T cells in the thymus (central tolerance) and the regulation of tolerance in mature T cells (peripheral tolerance). Advances in our understanding of the molecular basis for CD40 biology is based in two areas of research. First, a major breakthrough in our understanding of how CD40 transduces biological events centers on the identification of a novel protein that binds to the cytoplasmic tail of CD40 and may act as a signal transducing molecule. Secondly, advances in molecular modeling and mutagenesis of this ligand-receptor pair have helped to identify the critical receptor/ligand contacts in the gp39/CD40 complex. Advances in each of these areas are discussed.

Animals↗
PubMed1995

A template for generation and comparison of three-dimensional selectin models.

We have complemented multiple sequence alignments of the lectin domains of the selectins with an analysis of structurally invariant regions in X-ray structures of the mannose-binding protein (MBP) and E-selectin. The analysis shows that regions of structural conservation between MBP and E-selectin extend beyond regions of rigorous sequence conservation within the selectin family and suggests that reliable three-dimensional models of selectins from different species can be generated by modification of only a few backbone segments in E-selectin. A model of the L-selectin lectin domain is built and discussed with regard to observed differences in selectin specificity.

Amino Acid Sequence↗
PubMed1995

Cloning of the murine counterpart of the tumor-associated antigen H-L6: epitope mapping of the human and murine L6 antigens.

The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by mAb 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution of the single H-L6 residue Leu122 with Ser resulted in the H-L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Conformational similarity and systematic displacement of complementarity determining region loops in high resolution antibody x-ray structures.

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 A) of the essentially identical CDR loops become evident when the antibody beta-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought.

Amino Acid Sequence↗