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E A Padlan

Publications and source records attributed to E A Padlan.

At least 73 records · Page 4Linked to original sources
PubMed1988

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23).

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.

Animals↗
PubMed1988

Model-building study of the combining sites of two antibodies to alpha (1----6)dextran.

Models of the Fv portion (containing the variable regions of the heavy and light chains) of two monoclonal anti-alpha (1----6)dextran antibodies, W3129 and 19.1.2, were constructed from amino acid sequences and the known three-dimensional structures of the Fv portions of McPC603 and J539. The modeled combining site of W3129 has a protrusion on one side, formed by the long complementarity-determining region 1 of the light chain and the long complementarity-determining region 3 of the heavy chain, and has a cavity accommodating a glucose moiety. The model of the 19.1.2 site is basically flat with a shallow groove that can accommodate several internal glucose units. These results support the earlier conclusions, from ligand binding data, that W3129 has a cavity-type site, involving the terminal nonreducing glucose residue (endbinder), whereas 19.1.2 has a groove-type site.

Amino Acid Sequence↗
PubMed1987

Structure and refinement at 1.8 A resolution of the aspartic proteinase from Rhizopus chinensis.

The structure of rhizopuspepsin (EC 3.4.23.6), the aspartic proteinase from Rhizopus chinensis, has been refined to a crystallographic R-factor of 0.143 at 1.8 A resolution. The positions of 2417 protein atoms have been determined with a root-mean-square (r.m.s.) error of 0.12 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.010 A, and for angle distances it is 0.034 A. During the course of the refinement, a calcium ion and 373 water molecules, of which 17 are internal, have been located. The active aspartate residues, Asp35 and Asp218, are involved in similar hydrogen-bonding interactions with neighboring residues and with several water molecules. One water molecule is located between the two carboxyl groups of the catalytic aspartate residues in a tightly hydrogen-bonded position. The refinement resulted in an unambiguous interpretation of the highly mobile "flap", a beta-hairpin loop region that projects over the binding pocket. Large solvent channels are formed when the molecules pack in the crystal, exposing the binding pocket and making it easily accessible. Intermolecular contacts involve mainly solvent molecules and a few protein atoms. The three-dimensional structure of rhizopuspepsin closely resembles other aspartic proteinase structures. A detailed comparison with the structure of penicillopepsin showed striking similarities as well as subtle differences in the active site geometry and molecular packing.

Amino Acid Sequence↗
PubMed1987

Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action.

A peptide inhibitor, having the sequence D-His-Pro-Phe-His-Phe psi [CH2-NH]Phe-Val-Tyr, with a reduced bond between the two adjacent phenylalanines, has been diffused into crystals of the aspartic proteinase from Rhizopus chinensis (rhizopuspepsin, EC 3.4.23.6). X-ray diffraction data to 1.8-A resolution have been collected on the complex, which has been subjected to restrained least-squares refinement to an R-factor (R equals the sum of the absolute value of the difference between the observed and calculated structure factor amplitudes divided by the sum of the observed structure factor amplitudes) of 14.7%. The inhibitor lies within the major groove of the enzyme and is clearly defined with the exception of the amino-terminal D-histidine and the carboxyl-terminal tyrosine. The reduced peptide bond is located in the active site with close contacts to the two catalytic aspartyl groups. The active-site water molecule that is held between the two carboxyl groups is displaced by the inhibitor, as are a number of other water molecules seen in the binding groove of the native enzyme. A mechanism of action for this class of enzymes is proposed from these results.

Aspartic Acid Endopeptidases↗
PubMed1987

Three-dimensional structure of an antibody-antigen complex.

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.

Antibodies, Monoclonal↗
PubMed1986

Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A.

The crystal structure of the Fab of McPC603, a phosphocholine-binding mouse myeloma protein, has been refined at 2.7 A resolution by a combination of restrained least-squares refinement and molecular modeling. The overall structure remains as previously reported, with an elbow bend angle between the variable and constant modules of 133 degrees. Some adjustments have been made in the structure of the loops as a result of the refinement. The hypervariable loops are all visible in the electron density map with the exception of three residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of binding of the phosphate moiety of phosphocholine.

Amino Acid Sequence↗
PubMed1986

A model of the Fc of immunoglobulin E.

A model of the Fc of human IgE was constructed using the known three-dimensional structure of IgG Fc. On the basis of amino acid sequence homology, the CE2, CE3 and CE4 domains were modelled after CG3, CG2 and CG3, respectively. The mode of association of the CG2 and of the CG3 pairs of domains was assumed for the CE3 and the CE4 pairs, respectively; the CE2 pair of domains was assembled such that they interact like the CG3 domains. An asymmetric linkage is favored for the two inter-epsilon chain disulfide bridges, i.e. the bonds were assumed to be between non-homologous cysteines. The atomic interactions in the interface of the CE2:CE2 and CE4:CE4 domain pairs were computed, and predictions are made on the solvent accessibility of the individual residues in the fragment. The model can be useful in the study of regions that may be involved in the interaction of IgE with Fc(epsilon) receptors.

Amino Acid Sequence↗
PubMed1986

Localization of rabbit kappa light chain allotypic determinants.

The amino acid sequences of the constant regions of rabbit kappa light chains (C kappa) are remarkably divergent. The K1 allotypes differ at 47 of 106 positions; the K1 and K2 isotypes differ at three additional positions. Variability and structural dissimilarity plots reveal that most of these differences occur in clusters. Major hydrophilic areas are also found near some of these clusters. The structures of rabbit C kappa are modeled using the known alpha-carbon backbone structure of the Fab fragment of mouse myeloma protein McPC603. The effect of sequence variations upon the hypothetical three-dimensional structures was assessed and immunogenic determinants predicted and located. It was found that predicted determinants were external and located in or near loops. Two clusters of potentially interacting regions were predicted. Within each there could be several "topographical" and overlapping sets of epitopes that are recognized by different antibody-combining sites. One of the predicted immunogenic sites clearly interacts with the CH1 domain of the heavy chain. A heavy-chain dependent serological determinant has been correlated with amino acid differences in this region (kappa chain positions 121 and 124).

Amino Acid Sequence↗
PubMed1986

Antibody Fab assembly: the interface residues between CH1 and CL.

The effective assembly of an antibody molecule requires the proper association of the light and heavy chains, namely the tight, canonical association of VH with VL, and of CH1 with CL. In this paper the interaction of CH1 is examined by looking at the degree of conservation of residues in the interface between CH1 and CL, where CH1 can belong to any of the heavy chain classes, and CL can be either lambda or kappa. The three-dimensional structures of four antibody Fabs have been examined to see which are the significant interacting residues and to see whether they also correspond to the conserved residues in the different classes. It was found that there are a few hydrophobic residues buried in the interface which make numerous contacts with residues of the other chain and which remain invariant, or else are highly conserved. Around the periphery of the interface there are numerous interacting residues that have appreciable variability. Within the interface there is a cavity, the function of which may be to permit some changes in the central interface residues while still preserving the same relative orientation of CH1 and CL.

Amino Acid Sequence↗
PubMed1985

Refined crystal structure of deoxyhemoglobin S. I. Restrained least-squares refinement at 3.0-A resolution.

The crystal structure of deoxyhemoglobin S has been refined at 3.0-A resolution using the Hendrickson-Konnert restrained least-squares method. Comparison with the structure of deoxyhemoglobin A reveals a hingelike movement of the beta-chain A helices, which are involved in molecular contacts, toward the EF corners of their respective subunits. This movement brings the amino termini of the beta-chains closer to the molecular dyad. The A helices remain alpha-helical throughout their entire lengths. No other major structural difference is found between deoxyhemoglobin A and deoxyhemoglobin S.

2,3-Diphosphoglycerate↗
PubMed1985

Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal.

The refined crystal structure of deoxyhemoglobin S (Padlan, E. A., and Love, W. E. (1985) J. Biol. Chem. 260, 8272-8279) was used to analyze in detail the molecular interactions between hemoglobin tetramers in the crystal. The analysis confirms the close similarity and also the nonequivalence of the molecular interactions involving the two independent tetramers in the asymmetric unit of the crystal. The residue at the site of the hemoglobin S mutation, beta 6, is intimately involved in the lateral contacts between adjacent molecules. The molecular contacts in the crystals of deoxyhemoglobin S, deoxyhemoglobin A, and deoxyhemoglobin F were compared; some contacts involve the same regions of the molecule although the details of the interactions are very different. The effect of introducing an R state tetramer into the deoxyhemoglobin S strands was investigated using the known structure of carbon monoxyhemoglobin A. It was found that substituting a molecule of carbon monoxyhemoglobin A for one of the deoxyhemoglobin S tetramers results in extensive molecular interpenetration.

Carboxyhemoglobin↗
PubMed1985

Quantitation of the immunogenic potential of protein antigens.

A procedure to quantitate the immunogenic potential of protein antigens is presented. It is hypothesized that the intrinsic immunogenicity of an accessible region on the protein arises from the overall structural effect of the presence of the particular assemblage of amino acid residues in the given region. Structural parameters previously derived by Grantham [Science 185, 862-864 (1974)] to differentiate the various amino acids are assigned to each residue position. At each point chosen on the molecule, an av. value is computed due to all residues within 8.5 A of this point; it is proposed that this local average is proportional to the immunogenic potential of the region centered at this point. The method can be used to locate the immunodominant regions of a molecule and to compare the antigenicity of related molecules. Test calculations on hen egg-white lysozyme, sperm whale myoglobin and horse cytochrome c show that segments in these molecules, that have been shown in immunochemical studies to possess antigenic activity, are predicted by this method to be immunodominant.

Amino Acids↗
PubMed1984

Crystalline monoclonal antibody Fabs complexed to hen egg white lysozyme.

The Fab of a monoclonal anti-lysozyme antibody (HyHEL-10) has been crystallized as the free Fab and as the Fab-antigen complex. Crystals have also been grown of the antigen complex of the Fab of another monoclonal anti-lysozyme antibody (HyHEL-9), which recognizes a different binding surface of lysozyme. All three crystals diffract to at least 3 A resolution and are suitable for X-ray diffraction studies.

Animals↗
PubMed1984

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit.

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

Amino Acid Sequence↗
PubMed1979

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

An electron-density map of the mouse galactan-binding immunoglobulin J539 (IgA2,kappa) Fab has been calculated to a resolution of 4.5 A by the method of heavy atom isomorphous replacement with four derivatives. The map has been interpreted with the aid of a computer program which systematically searched for the best fit between the electron-density map and the known coordinates of individual immunoglobulin domains. The quaternary structure of J539 Fab at this resolution appears similar to that of another mouse immunoglobulin, IgA2,kappa Fab, McPC603. The model coordinates for J539 Fab should allow us to proceed directly to a high-resolution structure determination without further heavy atom isomorphous replacement.

Animals↗