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Biomedical subjects

E A Padlan

Publications and source records attributed to E A Padlan.

At least 19 recordsLinked to original sources
PubMed1992

Endogenous peptides of a soluble major histocompatibility complex class I molecule, H-2Lds: sequence motif, quantitative binding, and molecular modeling of the complex.

To gain insight into the rules that govern the binding of endogenous and viral peptides to a given major histocompatibility complex (MHC) class I molecule, we characterized the amino acid sequences of a set of self peptides bound by a soluble analogue of murine H-2Ld, H-2Lds. We tested corresponding synthetic peptides quantitatively for binding in several different assays, and built three-dimensional computer models of eight peptide/H-2Lds complexes, based on the crystallographic structure of the human HLA-B27/peptide complex. Comparison of primary and tertiary structures of bound self and antigenic peptides revealed that residues 2 and 9 were not only restricted in sequence and tolerant of conservative substitutions, but were spatially constrained in the three-dimensional models. The degree of sequence variability of specific residues in MHC-restricted peptides reflected the lack of structural constraint on those amino acids. Thus, amino acid residues that define a peptide motif represent side chains required or preferred for a close fit with the MHC class I heavy chain.

Amino Acid Sequence↗
PubMed1992

Structures of complexes of rhizopuspepsin with pepstatin and other statine-containing inhibitors.

The three-dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin-like peptide inhibitors of renin have been determined by X-ray diffraction methods and refined by restrained least-squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the "flap," a beta-hairpin loop region, that projects over the binding cleft and closes down over the inhibitor, excluding water molecules from the vicinity of the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp-35 and Asp-218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor.

Aspartic Acid Endopeptidases↗
PubMed1992

A modeling study of the alpha-subunit of human high-affinity receptor for immunoglobulin-E.

The extracellular portion of the alpha-subunit of human high-affinity receptor for immunoglobulin-E (IgE), which contains two immunoglobulin (Ig) domains, was modeled on the basis of sequence similarity with antibody domains of known three-dimensional structure. Each receptor domain contains 86 amino acid residues, and both domains were modeled as bilayer structures. In both domains, one layer is made up of three anti-parallel beta-strands and the other of four strands, with the two layers linked by a disulfide bridge. The two domains show significant sequence similarity with each other (22 identities) and with the homologous domains of the murine and rat high-affinity receptors for IgE and the Fc gamma receptors from various species. Two plausible modes of association of the domains were considered: In the first, the two domains were positioned end-to-end, with essentially only longitudinal interactions between them; in the second, the molecule is more bent, with more lateral interactions between the two domains. The models will be useful in the design of protein engineering studies of this and homologous receptors to delineate the site of interaction with ligand. Furthermore, they may lend themselves as possible probes in crystallographic analyses by molecular replacement techniques.

Amino Acid Sequence↗
PubMed1991

The nature and importance of the inter-epsilon chain disulfide bonds in human IgE.

IgE antibodies are best known for their pathological role in allergy. The class-specific effector sites are located in the epsilon chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite C epsilon 2 domains. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is unresolved. For structural and functional analysis site-specific mutations were introduced into the C epsilon 2 domain of recombinant human IgE. The introduction of an additional cyanogen bromide cleavage site (His246----Met) facilitated the identification of parallel disulfide bond pairing. This linkage was also confirmed for myeloma IgE PS by sequence determination of disulfide-linked C epsilon 2 dimers. Substitution of Cys241 and Cys328 by Ser does not destroy receptor binding, but reductive alkylation, or the replacement of Cys328 by Met, leads to loss of activity. This shows that covalent dimerization is not essential for IgE/receptor interaction and points to the importance of the structural integrity of the site surrounding Cys328, visualized in a new model of human Fc epsilon.

Amino Acid Sequence↗
PubMed1990

Binding characteristics of IgA 16.4.12E, a monoclonal antibody with specificity for the nonreducing terminal epitope of alpha-(1----6)-dextrans. Comparisons between IgA hybridoma 16.4.12E and myeloma W3129.

IgA 16.4.12E is a murine monoclonal antibody obtained following immunization with isomaltohexose linked to keyhole limpet hemocyanin. We have studied its interaction with methyl alpha-D-glucopyranoside and its derivatives bearing deoxy or deoxyfluoro groups, and with the methyl alpha-glycosides of a series of isomalto-oligosaccharides, some bearing deoxy or deoxy-fluoro groups at selected positions. From the data it is concluded that the antibody binds optimally to 4 sequential glucopyranosyl residues and that the protein subsite possessing the major affinity binds the terminal, nonreducing glucosyl group of that antigenic epitope. All the hydroxyl groups of that terminal glucosyl group are involved in hydrogen bonding, some in a donating and some in an accepting capacity. In the last part of the paper we report the construction of a possible model of the antibody, derived from its known amino acid sequence and the known crystalline structures of two closely related antibodies. It shows a pronounced cavity in the general immunoglobulin combining area which is flanked by 2 solvent-exposed tryptophanyl residues. A model recently reported for anti-dextran IgA W3129 shows a similar cavity with one such residue. Guided by hydrogen bonds, experimentally deduced from the comparison of the affinities of variously derivatized ligands, we suggest a speculative fitting for the nonreducing terminus of the dextran antigen, in the respective cavities of both IgA 16.4.12E and W3129.

Antibodies, Monoclonal↗
PubMed1990

Two Ig framework I epitopic specificities recognized by a rabbit antiserum and a monoclonal antibody to mouse VH chains.

The reactivity of 23 mouse monoclonal Ig with a rabbit polyclonal antiserum to VH of anti-alpha(1----6)dextran 19.22.1 and with a monoclonal anti-VH of anti-DNP MOPC315, when correlated with amino acid sequence, identified several residues in the first and third framework regions as being of potential importance in forming the epitope. Inhibition studies using synthetic peptides corresponding to residues 1-15 of the monoclonal Ig used to produce the poly- and monoclonal reagents provide evidence that the epitopes are predominantly, if not exclusively, specific for the N-terminal strand of the domain. Examination of known x-ray structures of mouse VH suggests that the primary difference between the two epitopes in the N-terminal strands is determined by the peptide chain structure due to Pro at position 9. Pro 9 appears essential for the epitope reactive with anti-VH MOPC315.

Amino Acid Sequence↗
PubMed1990

On the nature of antibody combining sites: unusual structural features that may confer on these sites an enhanced capacity for binding ligands.

A detailed analysis of the structural aspects of antibody-antigen interactions has been made possible by the availability of X-ray structures for three complexes of antilysozyme Fabs to lysozyme (reviewed by Davies et al.: J. Biol. Chem. 263:10541-10544, 1988.) Examination of the antigen-contacting residues in the three antilysozyme Fabs reveals the occurrence of a large number of aromatics, particularly tyrosines, and the absence of apolar, aliphatic residues. Calculation of the frequency of occurrence of the various amino acid types reveals that tyrosines are three times, and histidines and asparagines eight times, more likely to be found in the complementarity-determining regions than in the framework of the variable domains. Analysis of the solvent accessibility of the residues in Fvs (the modules containing variable domains of the light and heavy chains) of known three-dimensional structure indicates that tyrosines and tryptophans are more exposed when they occur in the complementarity-determining regions than when in the framework. Furthermore, many more of the asparagines in the complementarity-determining regions than in the framework are buried. These asparagines appear to have a structural role in that they hydrogen-bond through their side chains to other side chains and, even more so, to the protein backbone. The stabilizing effect of the asparagines, plus the rigidity of the framework, may serve to allow the greater exposure of the aromatic residues to solvent. In view of the greater potential contribution of aromatic side chains to the total binding energy, these results suggest that antibody combining sites have structural features that make them especially suited for interacting with ligands.

Amino Acids↗
PubMed1989

Structure of an antibody-antigen complex: crystal structure of the HyHEL-10 Fab-lysozyme complex.

The crystal structure of the complex of the anti-lysozyme HyHEL-10 Fab and hen egg white lysozyme has been determined to a nominal resolution of 3.0 A. The antigenic determinant (epitope) on the lysozyme is discontinuous, consisting of residues from four different regions of the linear sequence. It consists of the exposed residues of an alpha-helix together with surrounding amino acids. The epitope crosses the active-site cleft and includes a tryptophan located within this cleft. The combining site of the antibody is mostly flat with a protuberance made up of two tyrosines that penetrate the cleft. All six complementarity-determining regions of the Fab contribute at least one residue to the binding; one residue from the framework is also in contact with the lysozyme. The contacting residues on the antibody contain a disproportionate number of aromatic side chains. The antibody-antigen contact mainly involves hydrogen bonds and van der Waals interactions; there is one ion-pair interaction but it is weak.

Antibodies, Monoclonal↗
PubMed1988

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium.

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.

Amino Acid Sequence↗
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↗