Crystal Structure of Human Thioredoxin Oxidized by a Potential Anticancer Drug. Rogerio R. Sotelo-Mundo, Andrzej Weichsel, William R. Montfort, Department of Biochemistry, University of Arizona, Tucson AZ 85721

Thioredoxin is a small (~12 kDa) protein that regulates multiple biological processes through its oxidoreductase activity, and is found in all living cells. In addition to these intracellular functions, human thioredoxin is secreted by certain solid tumors, where it functions as an autocrine growth factor. We report here the 1.9 Å crystal structure of human thioredoxin after treatment with 1-methylpropyl 2-imidazoyl disulfide (IV-2), an inhibitor of the protein that displays antitumor activity in scid mice. Surprisingly, the structure displays an oxidized active site, but no other modifications. Subsequent experiments [1] suggest the reasons for this are two-fold: (1) IV-2 is a substrate for hTrx in much the same way as disulfides are in proteins, and (2) inhibition of hTrx by IV-2 occurs through modification of Cys 73, which is located away from the active site. We have previously shown that hTrx crystallizes as a homodimer covalently linked through a disulfide bond between Cys 73 of each monomer [2], and have recently determined the non-covalent dissociation constant for the protein in solution, which ranges from 6.1 uM at pH 3.8 to 165 uM at pH 8.0. That the covalent bond is not required for dimer formation was confirmed by the crystal structure of the Cys 73 -> Ser mutant [2]. In the present structure, obtained under different crystallization conditions, hTrx is also dimeric. Implications for the structural basis for inhibition by IV-2 will also be discussed.

[1] Kirkpatrick, D. L., Kuperus, M., Dowdeswell, M., Kunkel, M., Berggren, M., Angulo, M., and Powis, G. (1997) Biochem. Pharmacol., in press.

[2] Weichsel, A., Gasdaska, J., Powis, G., & Montfort, W. (1996) Structure 4: 735-751.