Abstract
Redox (reduction–oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol–disulphide exchange reactions between PDI and hVKORC1.
Highlights
Thioredoxins (Trxs) are disulphide reductases that are responsible for maintaining proteins in their reduced state inside cells
The models that were generated for the metastable states of human VKORC1 (hVKORC1) and their validation through in silico and in vitro screening have led to a conceptually plausible mechanism for enzymatic reactions based on a sequence array of hVKORC1-activated states involved in vitamin K transformation. These results suggest several additional questions, the most important being the real enzymatic machinery of hVKORC1 and its activation
Structures of protein disulphide isomerase (PDI) (PDB ID: 4ekz; [9]), ERp18 (PDB ID: 1sen; [15]) and Tmx1 (PDB ID: 1x5e; [5]) were used to extract the coordinates of a domain containing the CX1 X2 C motif (Table S1, Figure S1). This domain was chosen for the study of all proteins because ERp18, Tmx1 and Tmx4 proteins are only constituted of one Trx-fold domain a
Summary
Thioredoxins (Trxs) are disulphide reductases that are responsible for maintaining proteins in their reduced state inside cells. All membrane-associated Trx proteins possess an active site made up of two vicinal cysteine (C) residues embedded in a conserved CX1 X2 C motif These two cysteines, separated by two residues, play a key role in the transfer of two hydrogen atoms to the oxidised target and the breaking of the Trx–disulphide bond (Figure 1A). An intermediate state during the electron transfer is a mixed disulphide bond formed by a pair of cysteine residues from two proteins, which can be resolved by the nucleophilic attack of a thiol group from one of the flanking cysteine residues Through this mechanism, the disulphide is exchanged within one thiol oxidoreductase or between a disulphide donor and a target protein [3]. Thiol–disulphide exchange reactions occur between redox-sensitive biomolecules if donors and acceptors can interact in the appropriate orientations when attacking and leaving groups [4]
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