AbstractTo solve the inhibition mechanism of thymidylate synthase (TS) by N4-hydroxy-dCMP (N4-OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N4-OH-dCMP [crystal A, belonging to the space group C 1 2 1, with two monomers in asymmetric unit (ASU), measured to 1.75 Å resolution] or both N4-OH-dCMP and N5,10-methylenetetrahydrofolate (mTHF) (crystals B and C, both belonging to the space group C 2 2 21, each with a single monomer in ASU, measured to resolution of 1.35 Å and 1.17 Å, respectively). Whereas crystal A-based structure revealed the mTS-N4-OH-dCMP binary complex, as expected, crystals B- and C-based structures showed the enzyme to be involved in a ternary complex with N4-OH-dCMP and noncovalently bound dihydrofolate (DHF), instead of expected mTHF, suggesting the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of the one-carbon group to a hitherto unknown site and oxidation of THF to DHF. Moreover, both C(5) and C(6) inhibitor atoms showed sp3 hybridization, suggesting C(5) reduction, with no apparent indication of C(5) proton release. In accordance with our previous results, in all subunits of these structures the inhibitor molecule was identified as the anti rotamer of imino tautomer, forming, similar to deoxyuridine monophosphate, two hydrogen bonds with a conservative asparagine (mouse Asn220) side chain.

The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.

Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2 0 -deoxyuridine 5 0 -monophosphate (dUMP) and (3) 5-fluorodUMP (FdUMP) and N 5,10 -methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (''inactive'' and ''active,'' respectively) in the loop 181-197, in each of the three mTS structures, the loop 175-191, homologous to hTS loop 181-197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein's inability to adopt the inactive conformation. The binary structures of either dUMP-or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS-5-FdUMP-meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41-47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.