Protein crystal growth from biphasic system, structural and mechanistic studies of Escherichia coli and human thymidylate synthase, and crystal structure of human Sp17

Factors facilitating crystal nucleation and growth in biphasic systems, especially at the phase boundary are discussed. Biphasic systems of polyethylene glycol and ammonium sulfate were successfully used in obtaining crystals of tetragonal hen egg white lysozyme, which grew to half a millimeter in each dimension and diffracted to beyond 1.5 A resolution. The successful application of biphasic systems to produce high duality protein crystals makes them a useful alternative to monophasic experiments.;Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are: a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.;Human Sp17 is a protein that binds to sulfated carbohydrates of the extracellular matrix of the oocyte. We have recently obtained crystals of human Sp17 in two different forms: cubic and tetragonal, which both diffract X-rays to about 2.2 A resolution at a synchrotron source. The structure of one form, cubic, has been partially solved using SIRAS based on Yb3+ derivative. The crystals belong to the space group F432 and contain about 80% solvent. Inside crystals, molecules of Sp17 form an empty capsule with point group symmetry 432, built from 24 subunits, and stabilized by sulfate ions located on the three-fold axes. The same symmetry of oligomeric assemblies has been observed for ferritin and several viruses for which the capsule formation plays crucial physiological roles.