title: Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site.
abstract: The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees 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 upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight residues between 156 and 157 (h146-h153) are known to be alpha-helical in other eukaryotes, and lie close together on the outside of the protein in regions of 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 positioned to form a disulfide bond with the active site cysteine [cys 198 (h195)] in its unliganded conformation. The amino terminal 27 residues, unique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to substrate binding. The drug resistance mutation, Y6H, confers a 4-fold reduction in FdUMP affinity and 8-fold reduction in kcat for the dUMP reaction. Though indirectly connected to the active site, the structure suggests a mechanism of resistance that possibly involves a change in structure. This structure offers a unique opportunity for structure-based drug design aimed at the unliganded form of the human enzyme.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Schiffer
authors.forename: I J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: V J
authors.initials: VJ
authors.lastname: Davisson
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003461:Crystallography; D004251:DNA Transposable Elements; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005057:Eukaryotic Cells; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D017356:Synchrotrons; D013938:Thymidine Monophosphate / Q000096:biosynthesis; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D003856:Deoxyuracil Nucleotides; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase