Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala.

A pathological variant of human phosphoglycerate kinase, phosphoglycerate kinase-Uppsala, associated with chronic nonspherocytic hemolytic anemia has been found to differ from the normal enzyme by substitution of an arginine at position 206 (corresponding to position 203 in yeast) by a proline. In order to understand the structural and functional consequences of this mutation, the corresponding mutant in yeast phosphoglycerate kinase was constructed. The three-dimensional structure of this mutant was resolved at 2.9 A. Although the overall structure is not modified, small local changes were observed. The kinetic parameters of the mutant were not found to be greatly affected, the catalytic constant being lowered by only 10-20%. The most significant difference when compared with the wild-type enzyme is a decrease in stability by about 3 kcal/mol. The physiological implications of this instability are discussed.

Three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group.

The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2b kappa) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies. The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding.

X-ray diffraction studies of an anti-azophenylarsonate antibody and of an antigen-antibody complex.

X-ray crystallographic studies of the Fab fragments of two murine monoclonal antibodies of predefined specificity are under way. Diffracted X-ray intensities of the crystalline native Fab fragment of an anti-azophenylarsonate antibody and of three heavy atom derivatives have been measured to a resolution of 3.5 A. A preliminary 6-A resolution electron density map has been obtained. The 6-A resolution structure of an antigen-antibody (hen lysozyme-Fab) complex has been determined. There are close contacts between the antigen and the antibody over a large contact area, about 20 X 25 A. At least two segments of the polypeptide chain of lysozyme, of about 10 amino acids each (positions 19-27 and 116-129), are involved in the contacts, as well as all six complementarity-determining regions of the antibody. No gross conformational changes are observed in the antigen at this resolution, although there are some smaller local changes in areas in contact with the antibody and elsewhere. The effects of amino acid substitutions on antigen recognition by the monoclonal anti-hen lysozyme antibody were investigated using different, closely related lysozymes. These effects can be readily explained in terms of the three-dimensional model presented here. A 3.5-A resolution electron density map has been calculated and is currently under study.

Crystallization of the fab fragments of monoclonal anti-p-azophenylarsonate antibodies and their complexes with haptens.

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of Fab fragments from monoclonal anti-p-azophenylarsonate antibodies. Several crystalline forms were obtained with the Fab fragment from the R19.9 monoclonal antibody as well as with the complex between the hapten p-aminobenzenearsonic acid and Fab R19.9. The crystals of this hapten-Fab complex are similar to but not always isomorphous with the native Fab crystals. All the native and complex crystals were obtained using polyethylene glycol 6000 as crystallizing agent. Some of these crystalline forms diffract to a 2-A resolution or beyond and are suitable for high resolution x-ray diffraction analysis. A possible interpretation of hapten binding to crystalline Fab fragments from R19.9 and from the R9.3 monoclonal anti-p-azophenylarsonate antibody, implying conformational changes, is discussed.

Preliminary crystallographic study of the Fc fragment of a monoclonal IgG2b murine immunoglobulin.

The Fe fragment of a gamma 2b murine monoclonal anti-p-azophenylarsonate antibody (R19.9, IgG2b, kappa) has been crystallized. The crystals are tetragonal, space group P41 (or P43) with unit cell dimensions: a = b = 134.3 +/- 1.1 A, c = 144.0 +/- 0.7 A.