CRYSTAL-STRUCTURE OF HUMAN THYMIDYLATE SYNTHASE - A STRUCTURAL MECHANISM FOR GUIDING SUBSTRATES INTO THE ACTIVE-SITE

The crystal structure of human thymidylate synthase, a target for anti -cancer drugs, is determined to 3.0 Angstrom resolution and refined to a crystallographic residual of 17.8%. The structure implicates the en zyme in a mechanism for facilitating the docking of substrates into th e active site. This mechanism involves a twist of approximately 180 de grees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upo n substrate binding. The highly conserved, but eukaryote-specific inse rtion of twelve residues 90-101 (h117-128), and of eight residues betw een 156 and 157 (h146-h153) are known to be a-helical in other eukaryo tes, and lie close together on the outside of the protein in regions o f disordered electron density in this crystal form. Two cysteines [cys 202 (h199) and 213 (h210)] are close enough to form a disulfide bond within each subunit, and a third cysteine [cys 183 (h180)] is position ed to form a disulfide bond with the active site cysteine [cys 198 (h1 95)] in its unliganded conformation. The amino terminal 27 residues, u nique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to su bstrate binding. The drug resistance mutation, Y6H, confers a 4-fold r eduction in FdUMP affinity and an 8-fold reduction in k(cat) for the d UMP reaction. Though indirectly connected to the active site, the stru cture suggests a mechanism of resistance that possibly involves a chan ge in structure. This structure offers a unique opportunity for struct ure-based drug design aimed at the unliganded form of the human enzyme .