Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

Abstract

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

“…The presence of 50 mM peptide reduced the rate of consumption of CH 2 H 4 F Fluorescence studies to probe molecular mechanism of peptide inhibition Earlier structural and fluorescence studies with human TS established that the enzyme undergoes conformational switching between active and inactive states dependent on ligand binding. [26][27][28][29][30][31] The observation that the peptides were able to inhibit T. gondii TS-DHFR in an apo-specific manner, similar to what has been observed for human TS, raised the question of whether T. gondii TS might also utilize an analogous conformational switching to couple active and inactive conformational states. Conformational switching involves a change of position of a TS active site loop (residues 181-197 in human cor-responding to residues 475-491 in T. gondii), which contains the catalytic cysteine, Cys489, that would modulate transitions between active and inactive conformations ( Fig.…”

“…Earlier structural and fluorescence studies with human TS established that the enzyme undergoes conformational switching between active and inactive states dependent on ligand binding . The observation that the peptides were able to inhibit T. gondii TS–DHFR in an apo‐specific manner, similar to what has been observed for human TS, raised the question of whether T. gondii TS might also utilize an analogous conformational switching to couple active and inactive conformational states.…”

“…The TS domain from T. gondii undergoes conformational switching between active and inactive forms, the first reported nonprimate TS enzyme to do so . Previous studies have demonstrated that the residue Arg163 in human TS stabilizes its inactive conformation and is essential for conformational switching . The position of this residue corresponds to Asn457 in the TS domain of T. gondii TS–DHFR [Fig.…”

“…34 Previous studies have demonstrated that the residue Arg163 in human TS stabilizes its inactive conformation and is essential for conformational switching. [26][27][28][29]34 The position of this residue corresponds to Asn457 in the TS domain of T. gondii TS-DHFR [ Fig. 2(A)].…”

Analysis of mRNA recognition by human thymidylate synthase

“…The only de novo biosynthetic pathway to generate dTTP (2′-deoxythymidine-5′-triphosphate) requires reductive methylation of dUMP (2′-deoxyuridine-5′-monophosphate) to dTMP (2′-deoxythymidine-5′-monophosphate) by TS [ 1 ]. The TS enzyme is an obligatory homodimer [ 2 ] whose subunits associate with nanomolar affinity [ 3 ] to form a dimer that adopts an asymmetric conformation upon substrate binding [ 4 , 5 ]. Inhibition of TS leads to the cessation of DNA replication and thymineless death of proliferating cells [ 6 ], which renders the enzyme an attractive target for cancer chemotherapy [ 7 ].…”