The CXXC motif: crystal structure of an active-site variant of Escherichia coli thioredoxin

The 2.2 Å crystalline structure of an oxidized active-site variant of Escherichia coli thioredoxin (Trx) has been solved. Trx is a 12 kDa enzyme which catalyzes the oxidation of dithiols and the reduction and isomerization of disulfides in other proteins. Its active site contains the common structural motif CXXC. Protein-disulfide isomerase (PDI), a 57 kDa homolog of Trx, contains four Trx-like domains. The three-dimensional structure of PDI is unknown. PDI-deficient Saccharomyces cerevisiae are inviable. An active-site variant of Trx which complements PDI-deficient yeast has the active-site sequence Cys32-Val33-Trp34-Cys35 (CVWC). The reduction potential of oxidized CVWC Trx (E°′ = −0.230 V) is altered significantly from that of the wild-type enzyme (E°′ = −0.270 V). However, the structure of the oxidized CVWC enzyme is almost identical to that of wild-type Trx. The addition of valine and tryptophan in the active site is likely to increase the reduction potential, largely by decreasing the pK_a of the Cys32 thiol in the reduced enzyme. Unlike in wild-type Trx, significant protein–protein contacts occur in the crystal. Protein molecules related by a crystallographic twofold axis form a dimer in the crystal. The dimer forms as an extension of the twisted mixed β-sheet which composes the backbone of each Trx structure.