Crystal structure of a deletion mutant of human thymidylate synthase Δ (7–29) and its ternary complex with Tomudex and dUMP

Abstract

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS tenfold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

“…The Ca RMSD values between the liganded and unliganded mTS structures presented here, as well as between these structures and the crystal structures of other ternary complexes with dUMP and Tomudex, including those with hTS [15,16], rTS [17] and mTS [18], are shown in Table 3. The superimposition of the mTS, mTS-dUMP and mTS-FdUMP-meTHF structures is shown in Fig.…”

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

“…This region has not been reported to be involved in 5-FdUR resistance; structural analysis indicates that it is located on the active site pocket and interacts with folate-based inhibitors, providing a plausible rationale for resistance (1, 11-16). The N-terminal residues from Gly 5 through Gln 18 accumulated 10 substitutions; this segment is disordered in existing crystal structures and has no known function (11)(12)(13)(14)(15)(16). There is no obvious correlation between the frequency of either single or multiple substitutions observed in resistant mutants and the presence of ␤-sheets or ␣-helices.…”

“…Among these site-directed replacements is T51S, the most common mutation we have previously observed and present in the highly resistant variant T51S, G52S (6). Thr 51 participates in hydrogen bonding within the Arg 50 loop and with Val 313 after ligand-induced conformational changes of the C-terminal segment (1,(11)(12)(13)(14)(15)(16); 5-FdUR resistance could arise at Thr 51 from several mechanisms, including an effect on this conformational change. Two replacements at Asp 254 (D254N and D254E) were observed in multiply mutated variants (255, 318, 358), and we found that both, as well as the D254A substitution, conferred resistance as single mutations (Fig.…”

“…Last, the M219V replacement affects (8), form a hydrophobic collar around the p-aminobenzoic acid ring in the folate analog in a new ternary complex (16). A third group of 14 replacements was observed in the N-terminal 27 amino acids, which are disordered in crystal structures (1,(11)(12)(13)(14)(15)(16). This observation raises the possibility that at least some wild-type amino acids in the N-terminal region may have an unrecognized function(s) in 5-FdUR resistance, a possibility substantiated by identification of the single mutant E23G.…”

“…Although metabolically unstable, the P303L protein was nonetheless able to confer resistance against both 5-FdUR-and anti-folate-based TS inhibitors in transfected cells (10). Like Y33H, the P303L substitution is not in close proximity to the catalytic site as determined from crystal structures (11)(12)(13)(14)(15)(16).…”