STRUCTURES OF THYMIDYLATE SYNTHASE WITH A C-TERMINAL DELETION - ROLE OF THE C-TERMINUS IN ALIGNMENT OF 2'-DEOXYURIDINE 5'-MONOPHOSPHATE AND5,10-METHYLENETETRAHYDROFOLATE

Thymidylate synthase undergoes a major conformational change upon liga nd binding, where the carboxyl terminus displays the largest movement (approximately 4 angstrom). This movement from an ''open'' unliganded state to the ''closed'' complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The m utant lacking the C-terminal valine (V316Am) of the enzyme is inactive . X-ray crystal structures of V316Am and its complexes with dUMP, FdUM P, and both FdUMP and CH2H4-folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. Th e 2.7-angstrom binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are p ivoted approximately 20-degrees around the 3'-hydroxyl compared to dUM P in the wild-type enzyme. The 2.7-angstrom crystal structure of V316A m complexed with cofactor, CH2H4folate, and the substrate analog, FdUM P, shows these ligands bound in an open conformation similar to that o f the unliganded enzyme. In this ternary complex, the imidazolidine ri ng of the cofactor is open and has reacted with water to form 5-HOCH2H 4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex stru ctures open new venues for the design of novel TS inhibitors.