Biomedical subjects

J Bajorath

Publications and source records attributed to J Bajorath.

At least 91 records · Page 5Linked to original sources

PubMed1994

Immunoglobulin fold characteristics of B7-1 (CD80) and B7-2 (CD86).

B7-1 and B7-2 are expressed on antigen-presenting cells and bind to the CD28 and CTLA-4 receptors on T cells. These interactions trigger a costimulatory pathway that is essential for T-cell activation. B7-1 and B7-2 are members of the immunoglobulin superfamily (IgSF) and, despite sharing common function, have only limited sequence similarity. The B7-1 extracellular region was previously subdivided into 2 IgSF domains, an N-terminal V(ariable)-like domain, followed by a C(onstant)-like domain. We recently reported that the V-like domains of B7-1 and B7-2 share some significant sequence similarities with 3 major histocompatibility complex (MHC)-encoded members of the IgSF. We have now applied inverse folding methodology to assess the compatibility of the B7-1 and B7-2 extracellular region sequences with currently available 3-dimensional structures. In these calculations, the sequences of the N-terminal (V-like) domains in B7-1 and B7-2 were not compatible with known structures, including the IgSF V-set. In contrast, the sequences of the C-like domains were compatible with IgSF C-set structures and were best recognized by the beta 2-microglobulin (beta 2m) domain of MHC Class I. A sequence comparison of the C-like domains in the B7 molecules showed that 11 of 17 rigorously conserved residues in B7-1 and B7-2 are not IgSF C-1 set consensus residues. When mapped onto the corresponding positions of the beta 2m structure, the conserved residues in B7 cluster on the surface, where they may interact with the B7 V-like domain or other molecules.

Amino Acid Sequence↗

PubMed1994

Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors.

B7-0 or B7-2 (CD86) is a T cell costimulatory molecule that binds the same receptors (CD28 and CTLA-4) as B7-1 (CD80), but shares with it only approximately 25% sequence identity and is expressed earlier during an immune response. Here we show that human CD86 maintains similar (within approximately 2- to 3-fold) overall receptor binding and T cell costimulatory properties as CD80. However, CD80 and CD86 did not bind equivalently to CTLA-4: CD80 bound Y100A, a form of CTLA4lg with a mutation in the CDR3-like region, > 200-fold better than did CD86; inhibition of CD80-mediated cellular responses required approximately 100-fold lower CTLA4lg concentrations; and CD80-CTLA4lg complexes dissociated 5- to 8-fold more slowly, Thus, CD80 and CD86 utilize different binding determinants and have different kinetics of binding to CD28 and CTLA-4.

Abatacept↗

PubMed1994

Humanization of 60.3, an anti-CD18 antibody; importance of the L2 loop.

Monoclonal antibody 60.3 binds to the CD18 component of the beta 2 integrin family of adhesion molecules. 60.3 has potential clinical application in blocking the neutrophil-mediated organ damage which occurs following a myocardial infarct or hemorrhagic shock. Analysis of the nucleotide and deduced amino acid sequences of murine 60.3 shows that the light chain contains no amino acid substitutions relative to the closest germline sequence, while the heavy chain is heavily substituted. We report here the humanization of 60.3. The humanized antibody binds to CD18-bearing cells with approximately 4-fold less affinity than the murine or chimeric antibody. We have shown that modification of amino acid L50 in the L2 loop of the humanized antibody results in loss of binding, while modification of a structural determinant (H71) for the H2 loop has no effect.

Amino Acid Sequence↗

PubMed1994

Single-chain mono- and bispecific antibody derivatives with novel biological properties and antitumour activity from a COS cell transient expression system.

Single-chain antibody molecules were expressed from modified eukaryotic expression vectors as individual protein domains encoded on interchangeable cDNA cassettes. Two different single-chain antibody derivatives were constructed by linking individual light- and heavy-chain variable domains. The first was specific for the L6 tumour-associated antigen and the second was specific for human CD3. Each single-chain variable domain was genetically fused with an Fc 'tag' and expressed as a fusion protein in a COS cell transient transfection system. These single-chain antibody derivatives demonstrated specific binding to cells expressing appropriate antigen and bound with affinities similar to native antibody. The CD3 single chain molecule mediated stronger activation of PLC gamma 1 and similar levels of T-cell proliferation compared with native antibody. A bispecific Fv single-chain cassette was created by fusing the expression cassettes encoding the binding domains for L6 and CD3 single-chain molecules using oligonucleotide primers encoding a short 27-residue 'helical' peptide linker. The CD3-L6 variable domains were fused to the Fc tag and expressed in COS cells. The CD3-L6FvIg bispecific fusion protein mediated adhesion between T cells and L6-positive tumour cells, and stimulated potent T-cell proliferation and cytotoxicity against tumour cells expressing the L6 antigen.

Amino Acid Sequence↗

PubMed1993

Interaction of P-selectin (CD62) and its cellular ligand: analysis of critical residues.

P-Selectin (CD62, PADGEM, GMP140) is a membrane glycoprotein which is rapidly mobilized to the surface of activated platelets and endothelial cells where it mediates leukocyte-platelet and leukocyte-vascular endothelial cell adhesion, respectively. P-Selectin is a member of a family of adhesion molecules which includes the endothelial cell adhesion molecule E-selectin and the leukocyte adhesion molecule L-selectin. Selectins mediate cell-cell binding resulting from the interaction between the amino terminal lectin domains of the selectins and their respective carbohydrate ligands. Here we report on a three-dimensional model of the lectin domain of P-selectin which was derived on the basis of its structural homology to the rat mannose binding protein (MBP) whose crystal structure has recently been reported. On the basis of the model, a number of point mutants were prepared to identify the P-selectin binding site. The residues found to be important for binding are located in a shallow groove on the surface of the molecule composed of residues from the beta-2, -3, and -5 strands of the P-selectin lectin domain. A number of residues within this groove, which are conserved among all selectins, were found to be critical for P-selectin binding. They include Lys113, Tyr48, and Tyr94. The single substitutions Lys113Ala, Tyr48Ala, Tyr48Phe, Tyr94Ala, and Tyr94Phe abolished P-selectin binding to myeloid cells.

Amino Acid Sequence↗

PubMed1993

The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome.

The prominent role of the CD40 receptor in B cell responses led us to investigate the role of the gp39-CD40 interaction in a group of primary immunodeficient patients with defective antibody production. Here we report that patients with hyper-IgM syndrome (HIM) have a defective gp39-CD40 interaction. B cells from HIM patients express functional CD40, but their T cells do not bind CD40-Ig. These patients expressed normal levels of gp39 mRNA, but these mRNAs encode defective gp39 proteins owing to mutations in the extracellular domain of gp39. Soluble recombinant forms of gp39 containing these mutations were unable to bind CD40 and drive normal B cell proliferation. The gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for HIM had previously been mapped. These data suggest that a defect in gp39 is the basis of X-linked HIM.

Adolescent↗

PubMed1993

Knowledge-based model building of proteins: concepts and examples.

We describe how to build protein models from structural templates. Methods to identify structural similarities between proteins in cases of significant, moderate to low, or virtually absent sequence similarity are discussed. The detection and evaluation of structural relationships is emphasized as a central aspect of protein modeling, distinct from the more technical aspects of model building. Computational techniques to generate and complement comparative protein models are also reviewed. Two examples, P-selectin and gp39, are presented to illustrate the derivation of protein model structures and their use in experimental studies.

Amino Acid Sequence↗

PubMed1992

Similarity between fluorescein-specific T-cell receptor and antibody in chemical details of antigen recognition.

A computer-generated model of the single-chain variable V alpha V beta fragment of the RFL3.8 T-cell receptor (TCR) specific for fluorescein served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues. Selected backbone segments of the model representing regions of prominent sequence similarity between antibodies and TCRs were least-squares superimposed onto the corresponding segments of the crystallographically resolved 4-4-20 antibody complexed with its antigen, fluorescein. The superimposition placed the antibody-bound fluorescein molecule close to a cavity on the surface of the TCR model formed by the complementarity-determining region (CDR) loops. Some of the TCR cavity forming loops displayed sequence motifs related to canonical CDR loops previously found in antibodies. Six putative amino acid contacts were identified and single-chain TCRs with mutations at each of these positions were expressed in Escherichia coli, purified, refolded, and assayed for fluorescein binding. Five of the six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues are distributed among the beta 3, alpha 1, and alpha 2 CDR loops and show striking chemical similarity to the known fluorescein contact residues on 4-4-20. Thus, antibodies and TCRs are similar both in their overall architecture and in the chemical details of specific antigen recognition.

Amino Acid Sequence↗

PubMed1992

Chimeric L6 anti-tumor antibody. Genomic construction, expression, and characterization of the antigen binding site.

We report the cloning of the genomic variable region genes of the human carcinoma reactive murine monoclonal antibody L6 and their genetic linkage to human constant region exons to encode a human IgG1/kappa chimeric antibody. The chimeric protein was produced at levels greater than 20 micrograms/ml (enabling the initiation of clinical trials) and was found to have binding properties identical with that of the murine parent. The nucleic acid sequence of the variable regions was determined and found to be different than that previously reported (1). The deduced amino acid sequence was then used to generate a structural homology based three-dimensional model of the antibody binding site, which was found to share features with antibodies known to interact with a protein surface, but distinct from those that bind to carbohydrate epitopes. Biochemical analysis of binding between antibody and the in vitro-translated product of a cDNA clone that confers L6 immunoreactivity demonstrates that the antibody recognizes a protein epitope encoded by this transcript which requires the presence of membranes, but is unaffected by the removal of carbohydrate side chains.

Amino Acid Sequence↗

PubMed1991

Inhibition of proteinase K by methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone. An x-ray study at 2.2-A resolution.

The crystal structure of the transition state analog complex formed covalently between proteinase K and methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone was determined by x-ray diffraction methods at a resolution of 2.2 A and refined by constrained least squares to an R factor of 19.8% for the 11864 structure amplitudes greater than 1 sigma F. The chloromethyl ketone group is covalently linked with the active site functional groups His69(N epsilon) and Ser224(O gamma). The former has substituted for chlorine and the latter has attacked the carbon of the ketone group, thereby forming the tetrahedral carbon atom of the transition state analog. The peptide part of the inhibitor is in an extended conformation and fills subsites S1 to S5 of the substrate recognition site. Its backbone hydrogens bond with strands 100-104 and 132-136 of the substrate recognition site as the central strand of a three-stranded antiparallel beta-pleated sheet. This sheet formation is associated with a movement by approximately 1 A of strand 100-104 which is probably associated with the insertion of the bulky proline side chain. The methoxysuccinyl group is stacked on the phenolic side chain of Tyr104 that is a part of the bottom of the recognition site. Biochemical studies show that shorter inhibitors of this type are less effective than the longer one, because there are fewer hydrogen bonding and van der Waals/stacking interactions.

Amino Acid Chloromethyl Ketones↗

PubMed1991

Theoretical studies on the dihydrofolate reductase mechanism: electronic polarization of bound substrates.

We have applied local density functional theory, an ab initio quantum mechanical method, to study the shift in the spatial electron density of the substrate dihydrofolate that accompanies binding to the enzyme dihydrofolate reductase. The results shed light on fundamental electronic effects due to the enzyme that may contribute to catalysis. In particular, the enzyme induces a long-range polarization of the substrate that perturbs its electron density distribution in a specific and selective way in the vicinity of the bond that is reduced by the enzyme. Examination of the electron density changes that occur in folate reveals that a similar effect is seen but this time specifically at the bond that is reduced in this substrate. This suggests that the polarization effect may be implicated in the reaction mechanism and may play a role in determining the sequence whereby the 7,8-bond in folate is reduced first, followed by reduction of the 5,6-bond in the resulting dihydro compound.

Binding Sites↗

PubMed1991

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase.

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.

Binding Sites↗

PubMed1991

The electrostatic potential of Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of -10 electrons yet it binds ligands with net charges of -4 (NADPH) and -2 (folate or dihydrofolate). Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished. The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside "pockets" of positive potential that enable the enzyme to bind the negatively charged ligands. The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme. The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent-protein interface, is analogous to the situation occurring in superoxide dismutase. Thus DHFR is another case where the shape of the active site focuses electric fields out into solution. The positive electrostatic potential at the entrance of the ligand binding site in E. coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57--residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate. The latter has been postulated to be involved in the catalytic process. A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species. It is noted that, although the net charges of DHFRs from different species vary from +3 to -10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function.

Amino Acids↗

PubMed1991

Changes in the electron density of the cofactor NADPH on binding to E. coli dihydrofolate reductase.

Quantum-mechanical electron density calculations reveal that a significant polarization is induced in the cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate) on binding to the enzyme dihydrofolate reductase. The calculations indicate that electron density corresponding to approximately 0.7 electron charges is shifted within the molecule, extending over more than 20 A. Further calculations on proposed enzyme mutants show that the polarization of NADPH on binding to DHFR is, in large part, induced by a motif of three positively charged residues. This motif was also identified to be directly responsible for the positive electrostatic potential surrounding the cofactor binding site in the enzyme. The possibility of this long-range polarization of NADPH was originally proposed based on a previous study of ligand binding to DHFR where a conserved structural motif of three positively charged residues was found to play a major role in polarizing the substrate folate over its entire length of 18 A.

Arginine↗

PubMed1989

Long-range structural changes in proteinase K triggered by calcium ion removal.

The X-ray crystal structure of the subtilisin-type enzyme proteinase K at 1.5 A resolution shows that is has two binding sites for Ca2+. Scatchard analysis indicates that one Ca2+ binds tightly, with pK 7.6 x 10(-8) M-1, and the other only weakly. Although Ca2+ is not directly involved in the catalytic mechanism and is 16.6 A away from the alpha-carbon atoms of the catalytic triad Asp 39-His 69-Ser 224, the activity of proteinase K towards the synthetic substrate succinyl-Ala-Ala-Ala-p-nitroanilide drops slowly to approximately 20% of its original value when it is depleted of Ca2+. This is not due to autolysis of the enzyme. The X-ray crystal structure of Ca2+-free proteinase K shows that removal of Ca2+ from the tight binding site triggers a concerted domino-like movement of five peripheral loops and of two alpha-helices. At a distance of 25 A from this calcium-binding site, the geometry of both the secondary substrate binding site and of the catalytic triad is affected by this movement thereby reducing the activity of the enzyme.

Binding Sites↗

PubMed1988

The enzymatic activity of proteinase K is controlled by calcium.

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Ca1 is in near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C = O of Val16 and the two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6 +/- 2.5) x 10(-8) M associated with the tightly bound Ca1 whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Ca1 binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescence-spectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 degrees C to 46 degrees C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.

Calcium↗

PubMed1988

Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber.

The activity of proteinase K (EC 3.4.21.14), a subtilisin-related serine proteinase, was assayed with azoalbumin that showed non-expected behavior in substrate saturation curve because of interaction between albumin molecules. Succinyl-(Ala)n-p-nitroanilide with n = 2 and 3, yielded specific activities of 3.5, 13 units/mg protein, respectively, reflecting a chain length dependence. The influence of peptide chain length on binding to proteinase K was also observed using mono- and dipeptide chloromethyl ketone inhibitors. They showed a maximum inhibition. They showed a maximum inhibition of proteinase K in solution of only about 50% even at a more than 20-fold molar excess. With the above substrates, the Vmax is not affected in presence of 10, 20 and 30% methanol, but the Km differs remarkably, suggesting competitive inhibition. The activity of proteinase K shows a maximum at 37 degrees C, and a temperature profile with more than 80% maximum activity in the range 20-60 degrees C. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 mg/ml) in aqueous solution. It does not occur at higher proteinase K concentrations at or above 1.0 mg/ml, consistent with crystallographic studies.

Binding Sites↗

PubMed1988

X-ray and model-building studies on the specificity of the active site of proteinase K.

Proteinase K, the extracellular serine endopeptidase (E.C.3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl ketone to the active site of proteinase K was first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 A and 5.0 A resolution. The protein inhibitor complex was refined by restrained least-squares minimization with the data between 10.0 and 1.8 A. The final R factor was 19.1%, and the model contained 2,018 protein atoms, 28 inhibitor atoms, 125 water molecules, and two Ca2+ ions. The peptide portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.

Amino Acid Sequence↗