The dynamic disulphide relay of quiescin sulphydryl oxidase

Abstract

Protein stability, assembly, localization, and regulation often depend on formation of disulfide cross-links between cysteine side chains. Enzymes known as sulfhydryl oxidases catalyze de novo disulfide formation and initiate intra- and intermolecular dithiol/disulfide relays to deliver the disulfides to substrate proteins1,2. Quiescin sulfhydryl oxidase (QSOX) is a unique, multi-domain disulfide catalyst that is localized primarily to the Golgi apparatus and secreted fluids3 and has attracted attention due to its over-production in tumors4,5. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulfide formation pathways. How disulfides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. We determined the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulfide relay were found more than 40 Å apart in this structure, too far for direct disulfide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulfide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulfide bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented herein, reveals additional biochemical features that facilitate disulfide transfer in metazoan orthologs. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of novel catalytic relays.

Enzyme structure captures four cysteines aligned for disulfide relay

“…11 Nevertheless, the two molecules in the RnQSOX1 crystal asymmetric unit were found with the redox-active sites poised for interaction.…”

“…The structures of the oxidoreductase fragment of human QSOX1 and the MmQSOX1 Cys76Ala/Cys455Ser mutant [ Fig. 3(B)] both contained a water molecule in this position, 11 but the distance between the water and the C B sulfur in the former and the absence of the C B cysteine in the latter obscured the potential role of this water as a proton acceptor from the resolving cysteine. Other Trx-fold proteins, such as thioredoxin itself, contain buried acidic residues, which have been proposed to serve as proton transfer catalysts to the cysteine that resolves the mixed disulfide.…”

“…2), similar to the configuration of the previously determined structure of the Mus musculus QSOX1 (MmQSOX1) Cys76Ala/Cys455Ser mutant constrained by a disulfide bond between the two reactive cysteines Cys73 and Cys452. 11 The relative orientations of the oxidoreductase and sulfhydryl oxidase modules of the two RnQSOX1 molecules were roughly similar, differing by a rotation of about 3 , calculated as described (Materials and Methods).…”

“…Alternatively, attack by the C B from the accepting C A XXC B motif restores the initial conditions. It is possible to make the mixed disulfide resistant to resolution and capture it for observation by eliminating the C B cysteines, [6][7][8]10,11 but at the expense of losing key players in the reaction.…”

“…To prevent completion of the catalytic cycle, two mutations were introduced to trap the mixed disulfide between the Trx1 and Erv domains of QSOX enzymes. 11 Explicitly, the C B cysteines of the C A XXC B motifs were mutated to alanine or serine, leaving only the C A cysteine of each motif to form the interdomain disulfide. Structures of these species revealed how the QSOX oxidoreductase domain docks into a berth above the redox-active disulfide and FAD of the sulfhydryl oxidase module.…”