Structure determination of a monoclonal Fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.
Jel 42 is a monoclonal antibody specific for histidine-containing protein, a small phosphocarrier protein required for sugar transport in Escherichia coli. Fab fragments prepared from this antibody by papain digestion consisted of three major isoelectric forms which were separated on a chromatofocusing column. Two of these forms produced large crystals in space group P21 and unit cell dimensions a = 117.48 A, b = 66.56 A, c = 67.31 A, and beta = 118.7 degrees, with two Fab fragments per asymmetric unit. Data were collected to 3.5-A resolution. The structure of Fab Jel 42 was solved by the Molecular Replacement method (least-squares refined to R = 0.282) using the known structure of Fab HED 10 (12) as the search model; the amino acid residues of the hypervariable and elbow regions of Fab HED 10 were omitted from the starting model. A Fourier map calculated at this stage revealed electron density which corresponded to the hypervariable loops forming the antigen-binding crevice and the elbow region of Fab Jel 42. The elbow angles for the two independent Fab molecules are 159 and 167 degrees, similar to that of the Fab HED 10 search model which has an elbow angle of 162 degrees. There is no local noncrystallographic axis of symmetry relating the two molecules in the asymmetric unit.
Prasad
L
L
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Vandonselaar
M
M
Lee
J S
JS
Delbaere
L T
LT
D000911:Antibodies, Monoclonal / Q000032:analysis*; D001426:Bacterial Proteins; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D007525:Isoelectric Focusing; D008958:Models, Molecular; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr