The R163K Mutant of Human Thymidylate Synthase Is Stabilized in an Active Conformation: Structural Asymmetry and Reactivity of Cysteine 195

Abstract

Loop 181-197 of human thymidylate synthase (hTS) populates two conformational states. In the first state, Cys195, a residue crucial for catalytic activity, is in the active site (active conformer); in the other conformation, it is about 10 A away, outside the active site (inactive conformer). We have designed and expressed an hTS variant, R163K, in which the inactive conformation is destabilized. The activity of this mutant is 33% higher than that of wt hTS, suggesting that at least one-third of hTS populates the inactive conformer. Crystal structures of R163K in two different crystal forms, with six and two subunits per asymmetric part of the unit cells, have been determined. All subunits of this mutant are in the active conformation while wt hTS crystallizes as the inactive conformer in similar mother liquors. The structures show differences in the environment of catalytic Cys195, which correlate with Cys195 thiol reactivity, as judged by its oxidation state. Calculations show that the molecular electrostatic potential at Cys195 differs between the subunits of the dimer. One of the dimers is asymmetric with a phosphate ion bound in only one of the subunits. In the absence of the phosphate ion, that is in the inhibitor-free enzyme, the tip of loop 47-53 is about 11 A away from the active site.

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

“…0.36 diinactive protein fraction corresponding to a 60% abundance of the inactive form of hTS monomers, estimated from fluorescence data in this buffer (14,44), under the assumption that in the dimeric protein, the probability that a monomer be found in a particular conformation, whether active or inactive, is independent of the conformation of the other monomer. Furthermore, we did not observe a calorimetric signal when LR was titrated into an hTS sample that was presaturated with dUMP, consistent with the fact that dUMP is expected to convert all of the protein into the active form (44) (SI Appendix).…”

Replacement of Val3 in Human Thymidylate Synthase Affects Its Kinetic Properties and Intracellular Stability,

“…The high-AS structures of V3T, V3F, and V3L were solved using molecular replacement with the CNS software (22) using the hTS structure (1YPV) as the search model. The crystal structure of co-crystallized V3F was solved with the AMoRe software (23) from CCP4 suite of programs (24) using the R163K structure (PDB code 2RD8), which represents the active conformer (25), as the starting model. The crystal structures of V3L and V3Y at low-salt conditions were solved by molecular replacement to compensate for slightly different unit cell dimensions using the V3F structure as the starting model.…”

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

“…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.…”