Structures of thymidylate synthase with a C-terminal deletion: Role of the C-terminus in alignment of 2'-deoxyuridine 5'-monophosphate and 5,10-methylenetetrahydrofolate

Abstract

Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 A). 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 mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4folate 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. The 2.7-A binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20 degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-A crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 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 structures open new venues for the design of novel TS inhibitors.

Development and validation of a genetic algorithm for flexible docking 1 1Edited by F. E. Cohen

“…It is not clear why GOLD failed in the cases of 1NIS (Lauble et al, 1994), 1ROB (Lisgarten et al, 1993), 1TDB (Perry et al, 1993), 2AK3 (Diederichs & Schultz, 1991), 2MCP (Padlan et al, 1985) and 2MTH (Smith & Dodson, 1992). In 1TDB the predictions seemed very unstable, with hardly any consistency or clustering in the 20 binding modes produced by GOLD.…”

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

“…One conformational change that is known to occur involves TS catalysis: following ordered substrate binding (dUMP then CH 2 H 4 folate), the C terminus of TS closes over the substrates to create an active site cavity shielded from solvent (20 -25). It is thought that this is followed by the formation of an iminium ion involving the bridge methylene and N4 of CH 2 H 4 folate (25). We showed previously, using the wild-type enzyme, that there is a burst in consumption of the cofactor, CH 2 H 4 folate, at TS (26).…”

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

“…Lys28 of T4 CH is replaced by Arg23 in L.casei TS, for which a precise positioning of the folate is assisted by the C-terminus. More specifically, the C-terminal carboxylate of Val316 forms direct hydrogen bonds with Arg23 and Trp85, while Arg23 and the backbone carbonyl oxygen of Ala315 form direct and water-mediated hydrogen bonds to the folate cofactor (Perry et al, 1993;Carreras and Santi, 1995). These interactions involving the cofactor are not likely to be preserved in a T4 CH ternary complex, as a consequence of the substitution of this arginine with lysine as well as the much shortened C-terminus.…”

“…Due to the more open folate binding pocket in T4 CH, the remaining part of the cofactor, p-aminobenzoic acid ring and glutamate, may be less constrained by the T4 CH residues and may adopt multiple conformations in the ternary complex. The C-terminus plays a key role in positioning the folate cofactor in L.casei TS (Perry et al, 1993), which is critical for the hydride transfer from C-6 of the pterin moiety to the exocyclic methylene intermediate. In T4 CH, the pterin ring of CH 2 H 4 folate cofactor is expected to stack with the base of dCMP in a slightly altered fashion, as a subtle change in the positioning of the folate will be required to make the hydride transfer unfavorable without affecting the methylene transfer.…”