Background

Disulfide bonds are crucial for the activity and regulation of many proteins; therefore, disulfide bond formation and reduction is a critical aspect of cellular life. Members of the thioredoxin (Trx) superfamily use the nucleophilic cysteines of their “CXXC” active sites to facilitate the formation and reduction of disulfide bonds (Lu and Holmgren, 2014). Given their importance for cell survival, thioredoxin-domain proteins and the thioredoxin system have been proposed as drug targets against apicomplexan parasites (Becker et al., 2000; Krnajski et al., 2001; Andricopulo et al., 2006; Kehr et al., 2010; Biddau et al., 2018; Biddau and Sheiner, 2019; Cobb et al., 2021). Despite being essential and their potential as drug targets, the substrates of these Trx-domain proteins are often ill-defined, as it can be difficult to discern specific substrates from other interacting partners.

One strategy for identifying the substrates of Trx domains relies on the active site mode of action: the first cysteine of the “CXXC'' active site forms a mixed disulfide bond with a client protein, and the sulhydryl group of the second cysteine is used to resolve that bond. Mutagenesis of the second cysteine, often to alanine or serine, prevents the reduction of the bond between the Trx-domain and client protein, trapping the two proteins together (Jessop et al., 2007; Oka et al., 2013). Subsequently, mass spectrometry can be used to identify the substrate. A major disadvantage to this approach is that it requires modification to the active sites of endogenous enzymes, that may be essential, or exogenous expression of the mutated enzymes, both of which may prove lethal for the organism. Particularly in organisms like Plasmodium falciparum, which is haploid for the majority of its lifecycle and carries single copies of genes for essential Trx-domain proteins, this approach may prove especially difficult.

Given the challenges inherent to a genetic approach, activity based chemical crosslinking of Trx-domains to their substrates is a straightforward and powerful option for identifying those substrates. Divinyl sulfone (DVSF) is a bifunctional, electrophilic, and cell permeable crosslinker shown to have remarkable specificity for nucleophilic cysteines, like those within Trx-domain active sites (West et al., 2011). Treatment of cells with DVSF results in irreversible crosslinking between redox-active cysteines, and it has been used to trap and identify substrates of Trx-domain proteins and other redox-active enzymes in yeast and human cells (Naticchia et al., 2013; Allan et al., 2016; Araki et al., 2017). Demonstrating the power of this activity-based technique for identifying Trx-domain substrates in a non-model organism, we used DVSF to trap and identify substrates of Trx-domain proteins in the endoplasmic reticulum (ER) of P. falciparum parasites (Cobb et al., 2021). We also demonstrated its specificity for redox-active nucleophilic cysteines, as other cysteine-containing proteins did not react with DVSF, and further validated our results by demonstrating ablation of crosslinking, by mutation of reactive cysteine residues to alanine residues (Cobb et al., 2021). In this protocol, we describe the use of DVSF to trap substrates of ER-localized Trx-domain proteins in P. falciparum and the identification of these trapped substrates via mass spectroscopy. Given the robust nature of this protocol, it will be of use for similar experiments in diverse organisms and within multiple cellular compartments. Because this approach can be utilized with minimal genetic modification, requiring only the addition of an affinity tag to the Trx-domain protein if a suitable antibody is not otherwise available, this protocol will allow others working with difficult organisms to study the interactions between Trx-domains and their substrates.