Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Abstract

Loop 181-197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k cat /K m reduced by factors in a 2-12 range. One of the mutants, M190K, is however unique in having the value of k cat /K m smaller by a factor of~7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181-197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188-194 of this loop, assumes a different conformation with the positions of C a shifted up to 7.2 Å . This affects region 116-128, which became ordered in M190K while it is disordered in wt. The conformation of 116-128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.

Human thymidylate synthase with loop 181–197 stabilized in an inactive conformation: Ligand interactions, phosphorylation, and inhibition profiles

“…The conformation of insert 1 was ordered in M190K, with loop 181–197 trapped in an inactive conformation. In contrast, insert 1 in A191K is disordered, similar to that observed in crystal structures of hTS 4, 9…”

“…The catalytic activity of A191K is more than 25‐fold higher than M190K 9. Thus, the effect of temperature on the catalytic activity of A191K was analyzed (Fig.…”

“…Mutants were created to introduce charged residues at these sites. The resulting mutants, M190K and A191K, exhibited <1% and approximately 25% of the catalytic activity of hTS, with A191K exhibiting higher activity 9. The structure of eukaryotic‐specific insert 1, which is correlated with the conformation of loop 181–197, differed in crystal structures of the native proteins.…”

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.…”