PRIMARY AND TERTIARY STRUCTURES OF THE FAB FRAGMENT OF A MONOCLONAL ANTI-E-SELECTIN 7A9 ANTIBODY THAT INHIBITS NEUTROPHIL ATTACHMENT TO ENDOTHELIAL-CELLS

The murine monoclonal IgG1 antibody 7A9 binds specifically to the endo thelial leukocyte adhesion molecule-1 (E-selectin), inhibiting the att achment of neutrophils to endothelial cells. The primary and three-dim ensional structures of the Fab fragment of 7A9 are reported. The amino acid sequence was determined by automated Edman degradation analysis of proteolytic fragments of both the heavy and light chains of the Fab , The sequences of the two chains are consistent with that of the IgG1 class with an associated kappa light chain with two intrachain disulf ide bridges in each of the heavy and light chains. The tertiary struct ure of the antibody fragment was determined by x-ray crystallographic methods at 2.8 Angstrom resolution. The F(ab')(2) molecule, treated wi th dithiothreitol, crystallizes in the space group P2(1)2(1)2(1) with unit cell parameters a = 44.5 Angstrom, b = 83.8 Angstrom, and c 132.5 Angstrom with one Fab molecule in the asymmetric unit. The structure was solved by the molecular replacement method and subsequently refine d using simulated annealing followed by conventional least squares opt imization of the coordinates. The resulting model has reasonable stere ochemistry with an R factor of 0.195, The 7A9 Fab structure has an elb ow bend of 162 degrees and is remarkably similar to that of the monocl onal anti-intercellular adhesion molecule-1 (ICAM-1) antibody Fab frag ment. The 7A9 antigen combining site presents a groove resembling the structure of the anti-ICAM-1 antibody, and other antibodies raised aga inst surface receptors and peptides, Residues from the six complementa ry determining regions (CDRs) and framework residues form the floor an d walls of the groove that is approximately 22 Angstrom wide and 8 Ang strom deep and that is lined with many aromatic residues. The groove i s large enough to accommodate the loop between beta-strands beta(4) an d beta(5), of the lectin domain of E-selectin that has been implicated in neutrophil adhesion (1).