Human Thioredoxin Dimerization: Regulation by pH, Role of Asp60, and Crystal Structure of Asp60 to Asn Mutant. John F. Andersen and William R. Montfort, Department of Biochemistry, University of Arizona, Tucson, AZ 85721

The crystal structure of human thioredoxin revealed a covalent dimer in which monomers were connected through a disulfide bond and active sites were buried [1]. Since catalysis by thioredoxin occurs only for the monomeric protein, we wished to determine if the dimeric form occured in solution (perhaps for regulation) or was simply a crystallographic artifact. Several aspects of the structure were consistent with a functional dimer, including: a contiguous, largely hydrophobic dimer interface; high sequence homology for residues in the interface, and cystienes perfectly aligned for disulfide bond formtion. However, an Asp 60 - Asp 60 hydrogen bond also present in the dimer interface suggested the dimeric form might be unstable at physiological pH (crystals were grown at pH 3.8). To address this we used a gel-filtration technique to determine the apparent non-covalent dimer dissociation constants (K_app) for both the wild type protein and the mutant protein D60N at a variety of pHs. For the wild type protein, K_app varied from 6.1 uM at pH 3.8 to 165 uM at pH 8.0. The switch from the lower to higher value was apparently governed by a single titratable group with a pKa of 6.5, nearly three pH units above the usual pKa for aspartate. Dimerization of the D60N mutant protein was the same as wild type at low pH, but displayed almost no pH dependence, suggesting that (1) Asp 60 is reponsible for the increase in K_app at higher pH, and (2) that Asp 60 has a pKa of 6.5. The crystal structure of D60N was determined to a nominal resolution of 2.0 Å and, suprisingly, did not exhibit an Asn 60 - Asn 60 hydrogen bond. Rather, the two Asn 60 residues rotated away from one other, allowing four new water molecules to enter the dimer interface. Implications for human thioredoxin function as atumor-expressed growth factor will also be discussed.

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