Evolution of metamorphism in thymidylate synthases within the primate lineages.

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k(cat)) of mutant enzymes were 45-149% of hTS, with the lysine mutant (R163K) exhibiting the highest k(cat). The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K(d)) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.

Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions, phosphorylation, and inhibition profiles.

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E(act) and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.

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

Loop 181-197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180 degrees . 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 approximately 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(alpha) shifted up to 7.2 A. 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.