Preliminary crystallographic studies on human complement pro-factor D.

The recombinant zymogen of the human complement protein factor D has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 as precipitant. Two crystal forms obtained at pH 5.4 belong to space group P2(1). The crystals grow to dimensions of 0.6 mm x 0.3 mm x 0.3 mm in three days, are stable in the X-ray beam, and diffract to 2.4 A.

Mutational analysis of the substrate binding site of human complement factor D.

Complement factor D is a serine protease with a single natural substrate, C3b-complexed factor B, and very low catalytic activity against synthetic esters. The recently solved X-ray crystal structure of factor D has demonstrated certain key differences from other serine protease in the conformation of residues of the catalytic triad and the substrate-binding regions. To investigate possible contributions of unique amino acid substitutions to these distinct structural and functional features of factor D, we constructed a series of mutants by substituting trypsin substrate-binding residues for the corresponding factor D residues. Wild-type and seven mutant factor D cDNAs were expressed stably in Chinese hamster ovary cells, and the recombinant proteins were purified from culture supernatants and assayed by hemolytic, proteolytic, and esterolytic assays. The combined results indicate that residues Thr-198, Ser-199, Arg-202, and perhaps also Val-203 provide determinants for substrate binding and catalysis. The data also provide additional support for the hypothesis that the proteolytically active conformation of the active center of factor D is induced by its substrate, C3bB.