A three-dimensional model of an anti-lysozyme antibody.

The primary amino acid structure of the lysozyme-binding antibody, HyHEL-10, as determined by amino acid and nucleotide sequencing was utilized to construct a scale model of the Fv (variable region domain of immunoglobulin) using energy-minimized torsional angles of the McPC603 Fv as a prototype template. This model was in turn used as a template for generating a computer-built set of co-ordinates, which were subjected to a total of 600 steps of Adopted Basis Newton-Raphson energy minimizations using the program CHARMM. Only minimal shifts of the backbone (root-mean-square 0.76 A) were required to give an energetically stable structure with a favorable van der Waals' energy. Several notable features were evident from both the scale model and the energy-minimized computer model: (1) the shape of the antibody combining region is that of a very shallow concavity approximately 20 A X 25 A; (2) the concavity is acidic and non-hydrophobic and is bordered by hydrophobic segments; (3) the lower portion of the combining site is dominated by a cluster of tyrosine residues over the L3 and H2 areas; (4) a somatic mutation encoded by the J region of the heavy chain (JH) may contribute significantly to the complementarity of heavy chain H3 to the epitope on hen egg white lysozyme. In addition, the space-filling energy-minimized model revealed that residue 49L, a framework residue, was prominently exposed and accessible in the center of the combining-site concavity. The model suggests that variation in length of complementarity-determining regions may function not only to change directly the shape of the antibody combining site, but may also influence indirectly the nature of the antibody surface by changing the accessibility of residues not usually involved in antigen binding.

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen.

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.

Structure-function relationships of thrombin based on the computer-generated three-dimensional model of the B chain of bovine thrombin.

The advent of sophisticated computer graphics systems that permit the representation of macromolecular structure has made it possible to examine protein structure in detail. We have used one aspect of this technology to develop a model of thrombin. The model is based on structural and functional similarities this enzyme exhibits with respect to proteins found in the family of serine proteinases. This review has covered interpretations of the structure of the model based on analyses of data that had been collected before and after the model was developed. On one hand, the conceptualization of primary and secondary features in the model of the active site of thrombin has for the most part been preceded by data from experiments on the interaction of thrombin with naturally occurring substrates and inhibitors. The features of the model explain these data adequately. On the other hand, the model has been more recently used in an interactive way to derive information about the bioregulatory aspects of thrombin. The realization that the amino-terminus portion of the cyanogen-bromide fragment was probably not part of the chemotactic activity, because it was probably internalized in the native protein, has suggested that synthetic analogs should focus more on the carboxyterminus of the peptide. It is hoped that in the future the model will continue to serve more in this function and that it can be used to explore further other aspects about the structural and functional relationships of this enzyme.

A refined model for the variable domains (Fv) of the J539 beta(1,6)-D-galactan-binding immunoglobulin.

A refined protocol for building a hypothetical model of the J539 Fv is described. Computer programs for positioning amino acid side chains and structure energy minimization [CHARMM program of Brooks et al., J. comp. Chem. 4, 187-217 (1983)] were employed. Computer modeling was accomplished on an Evans and Sutherland picture system which permitted structure visualization in three dimensions. Peptide backbone breaksites were rejoined by monitoring for correct distances and torsion angles. A physical model was then constructed and used as a basis for further refinements such as aligning conformations around remodeled sites, adjusting proline substitutions and optimizing hydrogen-bond-forming potentials. This structure (J539-ADO) was energy minimized and the final coordinates were obtained from the energy-refined model. The resulting hypothetical J539 structure can be compared to the structure of J539 now being determined by X-ray crystallography. The procedures described can be used for other Fv fragments.

Rhodopsin's protein and carbohydrate structure: selected aspects.

A topographic model for rhodopsin has been constructed based upon evaluation of rhodopsin's sequence by a secondary structure prediction algorithm as well as chemical and enzymatic modification of rhodopsin in the membrane [Hargrave et al. (1983) Biophys. Struct. Mech. 9, 235-244]. The non-uniform distribution of several amino acids in the primary structure and within the topographic model is discussed. The seven predicted helices were evaluated and each helix was found to have one surface which is much more hydrophobic than the other. Stereoscopic views of a three dimensional model with a functional color-coding scheme incorporating these features are presented. The amino acid sequence of rhodopsin has been compared to other proteins in the Dayhoff Protein Data Bank. No obvious relationship to any other protein sequenced was found. High resolution proton magnetic resonance spectroscopy was used to reinvestigate the structure and relative proportions of rhodopsin's major and minor oligosaccharide chains. One major (Man3GlcNAc3) and two minor (Man4GlcNAc3 and Man5GlcNAc3) were observed.

Contributions of individual amino acid residues to the structural stability of cetacean myoglobins.

The acid-denaturation behavior of eleven cetacean myoglobins has been studied at two ionic strengths, 0.01 and 0.10 M, at 25.0 degrees C. The myoglobulins studied fall into four phylogenetic suborders, representing the sperm whales, dolphins, baleen whales, and beaked whales. The differences in response to acid denaturation among these closely related myoglobins are small but statistically significant. In three cases, free-energy differences between myoglobins can be ascribed to one amino acid difference and in three others to two differences. The differences in response were analyzed in terms of the changes in noncovalent interactions occurring in the native structure. The effects of changes in electrostatic interactions over the whole charge array were calculated for each myoglobin species by using the modified Tanford-Kirkwood theory. The predicted changes in stability correlated well with the experimental observations in most cases. When differences in hydrogen-bonding capability were considered at a first approximation, substantial effects were predicted. When these effects were taken in conjunction with the electrostatic interactions, the correlation with experiment was improved. Additionally, restrictions in motional freedom and packing constraints appeared to be significant in the single-site analysis. The detectable differences in stability due to single amino acid substitutions along with the small differences in stability between the cetacean suborders indicate that compensatory interactions provide the mechanism for the conservation of stability among the myoglobins studied.

Mapping epitopes on the insulin molecule using monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.

Spontaneous activation of a human proto-oncogene.

It has been recently shown that malignant activation of the c-has/bas proto-oncogene in T24 human bladder carcinoma cells was mediated by a single point mutation. A deoxyguanosine located at position 35 of the first exon of this proto-oncogene was substituted by thymidine. These findings predicted that the resulting oncogene would code for a structurally altered p21 protein containing valine instead of glycine as its 12th amino acid residue. We now report the spontaneous activation of the human c-has/bas proto-oncogene during transfection of NIH/3T3 cells. As in T24 cells, this in vitro activated oncogene also acquired malignant properties by a single point mutation. In this case we have detected a G leads to A transition, which occurred at the same position as the mutation responsible for the activation of the T24 oncogene. These results predict that the p21 protein coded for by the spontaneously activated c-has/bas gene will incorporate aspartic acid as its 12th amino acid residue. Computer analysis of the secondary structure of c-has/bas encoded p21 proteins indicates that substitution of the glycine residue located at position 12, not only by aspartic acid or valine but also by any other amino acid, would result in the same structural alteration. These findings indicate that a specific conformational change is sufficient to confer transforming properties to this p21 protein. Moreover, they predict that any mutation affecting the coding properties of the 12th codon of the c-has/bas proto-oncogene will lead to its malignant activation.

Molecular features and immunological properties of lactate dehydrogenase C4 isozymes from mouse and rat testes.

With the use of a computer graphics system, spacefilling models of mouse and rat testis lactate dehydrogenase C4 (LDH-C4) isozymes were constructed from amino acid sequence and published x-ray diffraction data. Thirty-two residues that differ between the mouse and rat LDH-C4 sequences are also displayed on the monomeric and tetrameric lactate dehydrogenase molecules. Immunological properties of mouse and rat LDH-C4 isozymes are compared and related to these 32 differences. Possible relationships between the structure, especially the amino acid sequence, and the unique enzymatic properties of LDH-C4 isozymes are also discussed.

Electronic distributions within protein phenylalanine aromatic rings are reflected by the three-dimensional oxygen atom environments.

The atomic environments of 170 phenylalanine-residue aromatic rings from 28 protein crystal structures are transformed into a common orientation and combined to calculate an average three-dimensional environment. The spatial distribution of atom types in this environment reveals a preferred interaction between oxygen atoms and the edge of the planar aromatic rings. From the difference in frequency of interaction of oxygen atoms with the edge and the top of the ring, an apparent net free energy difference of interaction favoring the edge of the ring is estimated to be about -1 kcal/mol (1 cal = 4.184 J). Ab initio quantum mechanical calculations, performed on a model consisting of benzene and formamide, indicate that the observed geometry is stabilized by a favorable enthalpic interaction. Although benzene rings are considered to be nonpolar, the electron distribution is a complex multipole with no net dipole moment. The observed interaction orientation frequencies demonstrate that these multipolar electron distributions, when occurring at the short distances encountered in densely packed protein molecules, are significant determinants of internal packing geometries.

Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases.

Computer-generated molecular models of the trypsin-like domains of blood coagulation factor IXa, Factor Xa, and thrombin have been prepared. These hypothetical models are based upon the sequence homology of the blood coagulation enzymes with the pancreatic serine proteases and the known three-dimensional structure of the pancreatic serine proteases. The internal structures and active sites of these enzymes are highly conserved. The high degree of substrate specificity which characterizes the blood coagulation enzymes appears to be defined not entirely by the active site, but by the unique molecular surface surrounding the active site of each enzyme. Several regions which demonstrate high sequence variability among these enzymes likely participate in forming the putative extended substrate binding sites.

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin.

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.

Mapping the antigenic epitope for a monoclonal antibody against lysozyme.

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of Köhler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.

A monoclonal antibody that defines an idiotope with two subsites in galactan-binding myeloma proteins.

An IgG1 monoclonal antibody HyX24-14 was derived from A/J mice that were immunized with the IgA XRPC24 (X24) galactan binding myeloma protein (GalBMP) of BALB/c origin by the Kohler-Milstein hybridoma technology. HyX24-14 specifically binds some but all GalBMP. Different patterns of binding using a panel of nine Gal BMP were found, depending upon the concentration of antibody and the antigenic target. From molecular models and amino acid sequence data, ti was proposed that the idiotope defined by HyX24-14 had two subsites, each of which appeared to be able to bind independently to the antibody.