CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION ANALYSIS OF DISCOIDIN I. Yuri D. Lobsanov¹, Jung-Kay Chiu¹, Chi-Hung Siu² and James M. Rini¹. Departments of Molecular and Medical Genetics and Biochemistry¹, Banting and Best Department of Medical Research and Department of Biochemistry², University of Toronto, Toronto, Ontario, M5S 1A8 Canada

Discoidin I is a (-galactoside binding lectin involved in Dictyostelium discoideum cell adhesion. Starvation of slime mold amoebae results in the expression of the lectin and the formation of fruiting bodies. Discoidin I is physiologically active as a tetramer. The discoidin domain is a protein module which has recently been identified on two proteins in the coagulation cascade (Factor V and VIII), as well as cell surface molecules, including the neural antigen A5, thought to be involved in retinal axon targeting, and the tyrosine kinase signalling receptors DDR, Ptk-3 and Tyro 10.

Discoidin I was purified from slime mold culture by affinity chromatography on Sepharose-4B. Protein was eluted by 0.3 M galactose in 20 mM Tris(HCl pH 7.2, 1mM EDTA and 150 mM NaCl. Crystals have been obtained by the hanging-drop vapor-diffusion technique. The well solution contains 1.8 M ammonium sulfate, 100 mM MES pH 6.5 and 10 mM CoCl₂. The protein-sugar mixture contains 10 mg/ml protein and 100 mM thio-di-galactoside in 20 mM Tris(HCl pH 7.2 and 1mM EDTA. Rod-like crystals reach dimensions of 0.2 x 0.2 x 0.8 mm within 1 - 4 weeks and diffract to at least 2.4 on a conventional rotating anode. The crystals grow in a trigonal space group (a=66.0 , b=66.0 , c=149.5 ), with one molecule per asymmetric unit, yielding a solvent content of 63.5%. Native data to 2.6 resolution have been collected and heavy atom screening is in progress.