STRUCTURAL BASIS OF CHEMOTHERAPY RESISTANCE MEDIATED BY THYMIDYLATE SYNTHASE Peter H. Sayre, Carleton R. Sage, Robert M. Stroud, Dept. of Biochemistry & Biophysics, University of California, San Francisco 94143

The structure of a mutant thymidylate synthase (TS) representative of a chemotherapy-resistant enzyme reveals distinct atomic shifts away from the active site of absolutely conserved residues critical for ligand binding.

TS inhibition is an important mechanism for the chemotherapy agent 5-fluorouracil (5-FU), which prolongs life in patients with stage III colon cancer. Cells containing a well-characterized mutant form of the human enzyme (Tyr 33 -> His) exhibit relative resistance to the 5-FU metabolite 5-fluoro-2'-deoxyuridine. To investigate the structural correlates of this chemotherapy resistance, we took advantage of the extensive collection of wild type and mutant structures of E.coli TS built so far in this laboratory and used site-directed mutagenesis to generate the corresponding mutation in E. coli TS (Tyr 4 -> His). The Tyr 4 -> His mutant E. coli TS was expressed in bacteria, purified by ion exchange chromatography and crystallized alone or in the presence of substrate and cofactor ligands. The structure of the mutant molecule was refined to a crystallographic R factor of 22% (R_free = 26%). Differences between mutant and wild type were also studied by examination of F_o - F_o difference maps.

Estimates of coordinate error yielded expected differences from true atomic positions of 0.35 - 0.4 Å. F_o - F_o difference maps allowed smaller changes between mutant and wild type structures to be discerned. These differences are concentrated in the region between the N-terminal A helix and the large central active site. The difference maps clearly reveal the loss of electron density around the position of the tyrosine hydroxyl group absent in the mutant enzyme. The hydrogen-bonding network linking the N-terminal A helix to the J helix that intervenes between the A helix and active site cavity is disrupted, since the Tyr 4 hydroxyl is no longer available to interact with side chain carbonyl of Val 170. A concerted movement of atoms away from the active site toward the mutant histidine residue propagates across the base of the J helix. The distinct atomic shifts of invariant Asp 169 away from the active site may prevent interactions between its main chain amide and the pyrimidine of the 5-fluoro-2'-deoxyuridylate inhibitor and between the Asp 169 side chain and the quinazoline ring of the cofactor. These movements could explain the decreased sensitivity of the mutant enzyme to 5-fluoro-2'-deoxyuridylate inhibition.