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

Abstract

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.

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

“…Analyses of crystal structures of mTS bound with N 4 -OH-dCMP suggest that the molecular mechanism of an apparent strong preference for the anti rotamer of the imino inhibitor form, described in [43], is connected with the His190 presence. It was confirmed by both crystallographic studies [16,44] and molecular dynamics simulations [27] that the presence of His190 causes a steric hindrance preventing the syn form from being bound (Figure 9).…”

“…Electrostatic potential computations for the crystal structures of mouse thymidylate synthase alone (mTS, PDB ID: 3IHI) [ 44 ] and in binary complexes with dUMP (PDB ID: 4E5O) [ 44 ] and N OH-dCMP (PDB ID: 4EIN) [ 16 ] have been performed with continuum electrostatic calculations using Adaptive Poisson–Boltzmann Solver (APBS) v. 1.5 [ 87 , 88 ]. First, the crystal structures were prepared for APBS computations with PDB2PQR v. 2.1.1 [ 89 ], enabling the estimation of titration states and adequate protonation of molecules and the generation of pqr files with atomic charges and radii.…”

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies

“…This is in contrast to the TS-catalyzed reaction with dUMP or FdUMP and meTHF when the imidazolidine ring opens at the N(10) site, and the methylene group linking the meTHF N (5) and N(10) atoms, remains bound to the N(5) atom. The resulting N(5)=C( 11)H 2 group is responsible next for the electrophilic attack on the pyrimidine ring C(5) of the substrate/analog [13]. Thus, with N 4 -OH-dCMP bound in the active site the imidazolidine ring opens at the N(5) site, in a striking contrast to the corresponding phenomenon observed with dUMP and FdUMP, the difference resulting apparently from the presence of the N 4 -OH residue.…”

“…The lack of clearly defined electron density for the final few (four in this case) residues of the C-terminus of subunit B has been observed previously for several structures of TS complexes, e.g., rat TS-dUMP-Tomudex (PDB ID: 1RTS and 2TSR [17]), CeTS-dUMP-Tomudex (PDB ID: 5NOO [18]), and mTS-dUMP-Tomudex (PDB ID: 4EB4 [19]). Spatial orientation of His190 present in the 4EZ8 structure (or of the corresponding histidine residue in any other specific variant of TS) is also found in multiple other structures of the mouse enzyme, e.g., 4EIN (mTS-N 4 -OH-dCMP complex [14]), 4E5O (mTS-dUMP [20]), 6F6Z (mTS-N 4 -OH-dCMP soaked with meTHF), 4EB4 (mTS-dUMP-Tomudex [19]), 3IHI (mTS apoenzyme [13]), and as one of two alternative conformations in 5BY6 (TspTS-dUMP [18,21]). On the other hand, an alternative orientation of His190 present in the 5M4Z structure is also observed in several other structures, including 5BY6 (as the other alternative conformation with 0.6 occupancy), 4PSG (CeTS-N 4 -OH-dCMP complex), 5NOO (CeTS-dUMP-Tomudex [18]), 5FCT (mTS-FdUMP-meTHF [13]), 4IRR (CeTS-dUMP [19]), 4IQB (CeTS apoenzyme [18]), and 4ISW (phosphorylated CeTS-dUMP [22]).…”

“…In accord, N 4 -OH-dCMP remained bound to the enzyme under conditions of the SDS electrophoresis [15]. Of note is that in a ternary mTS complex, formed in the presence of FdUMP and meTHF (PDB ID: 5FCT; Table 1), all three components were covalently bound [13], as expected based on the corresponding TS structures published previously [12]. The latter proves meTHF to be stable under crystallization conditions, thus confirming that the previously mentioned structure (PDB ID: 4EZ8) is not an artifact.…”