The dynamic disulphide relay of quiescin sulphydryl oxidase

Abstract

Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides 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. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.

Figure 1. TbQSOX undergoes domain re-orientation to accomplish disulfide relay

a, CXXC motifs are illustrated as pairs of yellow balls in maps of trypanosome and mammalian QSOX enzymes. Other cysteines are shown in Supplementary Fig. 13. Fused hexagons represent the FAD cofactor. A degenerate Erv-like domain is designated “ψErv”. Arrows depict the electron transfer relay from reduced substrates to molecular oxygen and the corresponding outward flow of disulfide equivalents from QSOX to its substrates. b, TbQSOX structure colored according to a. Disulfides in CXXC motifs are in space filling representation (Cβ atom green, sulfur yellow). Structure stereo views are in Supplementary Fig. 14. c, Color-coded intramolecular distances between fluorophore-labeled TbQSOX cysteines and the bound FAD cofactor. d, Structure of TbQSOX_C. e, Surface representation of TbQSOX_C and zoom into the interdomain interface. FAD is in space-filling representation with oxygen atoms (red) labeled. Structure figures were made using PyMOL (http://www.pymol.org).

Figure 2. Changes in the TbQSOX conformational ensemble during catalysis

a, Fluorescence of Pacific Blue conjugated at position 160 in resting TbQSOX (blue), TbQSOX oxidizing the model substrate dithiothreitol (DTT) (red), and upon oxygen depletion and conversion of the energy acceptor FAD to FADH₂ (black). Inset, surface representation of TbQSOX_C, as in Fig. 1e, with FAD and donor labeling site indicated. b, Kinetics of donor fluorescence during TbQSOX oxidation of DTT. Upon DTT injection, labeled enzyme rapidly converts to a state showing greater FRET efficiency (upper right panel). Other panels show longer time scales at various initial DTT concentrations. At starting DTT above ~200 μM (left panel), oxygen becomes limiting, the flavin becomes trapped as FADH₂ (see Fig. 1a), flavin absorbance at ~450 nm drops dramatically, and donor fluorescence increases correspondingly. At starting DTT concentrations of 200 μM or below (bottom right panel), DTT is limiting, and the enzyme returns to its oxidized, resting state.

Figure 3. Mammalian QSOX and mechanistic insights into the QSOX catalytic cycle

a, The structures of HsQSOX1(PDI) (left) and HsQSOX1(Erv). Disulfides in CXXC motifs are shown. Other cysteines appear in Supplementary Fig. 13. b, Structure of MmQSOX1_C. c, Comparison of HsQSOX1(PDI) (left) with MmQSOX1_C (right) shows rearrangements in the Trx1 redox-active region upon formation of the disulfide-transfer intermediate. Interatomic distances (dashed lines) are in Angstrom. d, Tethering increases the effective concentration of the HsQSOX1 disulfide transferring module. Initial oxygen consumption rates were recorded for 100 nM HsQSOX1(Erv) and varying concentrations of HsQSOX1(PDI) (filled circles) or thioredoxin (filled squares) upon injection of DTT. Gray, dashed line indicates the turnover number for intact HsQSOX1, measured at 100 nM. By extrapolation, ~250 μM HsQSOX1(PDI) with 100 nM HsQSOX1(Erv) would be expected to support a similar reaction rate (gray arrow). e, Summary of the structural basis of the QSOX catalytic mechanism, as revealed in this study. Adjacent yellow balls with black bar indicate disulfide bonds; separated yellow balls indicate reduced thiolates. The “+” symbols in the closed conformation (lower right) represent arginine side chains from either the Trx1 domain (in TbQSOX) or the Erv domain (in TbQSOX and mammalian QSOX1) that may contribute to the electron-withdrawing ability of the FAD. The “backstop” represents conserved Erv domain loops (Supplementary Fig. 9).