Mechanism-based kinetic trapping can be applied to human Trx1
To determine whether kinetic trapping can be applied to human Trx1, we created recombinant wild-type and mutant Trx proteins, each equipped with a C-terminal dual affinity tag composed of a streptavidin-binding peptide (SBP) and a hexahistidine tag. To create a trapping mutant, the second cysteine of the 32CXXC35 motif was replaced by serine (C35S). Trx1 harbors three additional cysteine residues distal to the active site (cysteines 62, 69 and 73). As these residues are dispensable for catalytic activity but may cause oxidative inactivation by either intra- or intermolecular disulfide bond formation (Casagrande et al, 2002; Watson et al, 2003), we also created mutants containing amino-acid substitutions for those additional cysteines (Figure 1B; CCCCC, CCAAA, CSCCC, CSAAA and SSAAA annotate the identity of residues 32, 35, 62, 69 and 73).
To test whether Trx1(C35S)-based trapping is capable of identifying known Trx1 target proteins, Trx1(CSAAA) was allowed to react with cytosolic proteins released from digitonin-permeabilized cells. Incubation led to the formation of a reproducible pattern of distinct mixed disulfide conjugates as visualized by silver staining of a SDS–PAGE gel under non-reducing conditions (Figure 1C, lane 7). In accordance with the trapping mechanism, conjugation strictly depended on the availability of the N-terminal thiol (Cys-32) and the concurrent absence of the C-terminal thiol (Cys-35), as wild-type or cysteine-free Trx1 did not capture any proteins (Figure 1C, lanes 3, 5 and 9). The pattern of trapped proteins was not significantly influenced by the presence or absence of the non-catalytic cysteines (data not shown). The main cytosolic interaction partner of Trx1(CSAAA) was identified as peroxiredoxin-1 (Prx1) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The Trx1–Prx1 association was further confirmed by immunoblotting (data not shown). The Trx1–Prx1 disulfide-linked conjugate is maintained under non-reducing conditions (Figure 1C, lane 7) and cleaved into its monomer constituents under reducing conditions (Figure 1C, lane 8).
To test whether Trx1(CSAAA) would also undergo authentic interactions under conditions more typical of an extracellular environment, we allowed Trx1(CSAAA) to react with human plasma proteins. To avoid nonspecific absorbance to high-abundance serum proteins, we applied Trx1(CSAAA) to a <30 kDa plasma ultrafiltrate, leading to the capture of a small number of proteins, as visualized by colloidal Coomassie staining (Figure 1D). Using LC-MS/MS, the principal interaction partner from the plasma ultrafiltrate was identified as peroxiredoxin-2 (Prx2), a well-established target protein of Trx1, recently found to be present in human plasma (Chen et al, 2004). These experiments provided proof-of-principle evidence that kinetic trapping is capable of capturing and identifying proven target proteins of human Trx1 from both intra- and extracellular environments.