1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark
Thioredoxin (Trx) is an ubiquitous protein disulfide reductase that possesses two redox active cysteines in the conserved active site sequence motif, Trp-CysN-Gly/Pro-Pro-CysC situated in the so called Trx-fold. The lack of insight into the protein substrate recognition mechanism of Trx has to date been a hindrance for comprehensive understanding of the functions and biological roles of Trx. This issue is addressed in the present study on two h-type Trxs, HvTrxh1 and HvTrxh2 from barley seeds. The crystal structure was determined for a stable, disulfide-linked complex of a HvTrxh2 mutant (Cys49Ser) and a mutant of an in vitro substrate alpha-amylase/subtilisin inhibitor (BASI) (Cys144Ser), as a reaction intermediate-mimic of Trx-catalyzed disulfide reduction. The resultant structure showed a sequence of BASI residues along a conserved hydrophobic groove constituted of three loop segments on HvTrxh2 surface, associated through several van der Waals contacts and three backbone-backbone hydrogen bonds resembling beta-sheet formation. Moreover, a pattern of interactions essentially identical to that in HvTrxh2-S-S-BASI was observed in the structure of HvTrxh1 crystallized in the oxidized form. In the crystal lattice, HvTrxh1 formed a dimer, in which a loop segment from one molecule was situated along the hydrophobic groove at the active site of another molecule. The observed manner of protein recognition by Trx was similar in the central part to the glutathione recognition mechanisms of Trx-fold proteins glutaredoxin and glutathione transferase. This study suggests that the features of main chain conformation as well as charge property around disulfide bonds in protein substrates are important factors for interaction with Trx. Moreover, this study describes a detailed structural and biophysical comparison of the two coexisting h-type barley Trxs, HvTrxh1 and HvTrxh2.