Vitamin K epoxide reductase prefers ER membrane- anchored thioredoxin-like redox partners Sol Schulman, Belinda Wang, Weikai Li, and Tom A. Rapoport1 Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 Contributed by Tom A. Rapoport, July 11, 2010 (sent for review May 17, 2010) Vitamin K epoxide reductase (VKOR) sustains blood coagulation of this reaction (9). In a first step (Fig. 1A), the reduced CXXC by reducing vitamin K epoxide to the hydroquinone, an essential motif of the Trx-like protein domain transfers electrons to two cofactor for the γ-glutamyl carboxylation of many clotting factors. conserved cysteines in a periplasmic loop of VKOR (Fig. S1); this The physiological redox partner of VKOR remains uncertain, but is loop is located between transmembrane (TM) segments 1 and 2 likely a thioredoxin-like protein. Here, we demonstrate that human of the four-TM bundle surrounding the quinone. The reaction VKOR has the same membrane topology as the enzyme from proceeds through a mixed disulfide bridge intermediate, in which Synechococcus sp., whose crystal structure was recently deter- the N-terminal cysteine in the CXXC motif of the Trx-like mined. Our results suggest that, during the redox reaction, domain is linked with the N-terminal loop cysteine in VKOR Cys43 in a luminal loop of human VKOR forms a transient disulfide (Fig. 1A; boxed intermediate). This mixed disulfide bridge is sub- bond with a thioredoxin (Trx)-like protein located in the lumen of sequently resolved by attack of the C-terminal cysteine in the the endoplasmic reticulum (ER). We screened for redox partners of CXXC motif of the Trx-like domain, resulting in its oxidation with VKOR among the large number of mammalian Trx-like ER proteins concomitant reduction of the VKOR loop cysteines. In the next by testing a panel of these candidates for their ability to form this step, the electrons are transferred from the loop cysteines to a specific disulfide bond with human VKOR. Our results show that CXXC motif at the periplasmic edge of the fourth TM of VKOR VKOR interacts strongly with TMX, an ER membrane-anchored (Fig. 1A). Finally, the disulfide bridge in the CXXC motif Trx-like protein with a unique CPAC active site. Weaker interactions of VKOR is regenerated by transfer of electrons to vitamin K. were observed with TMX4, a close relative of TMX, and ERp18, the The hydroquinone leaves into the lipid phase and is reoxidized smallest Trx-like protein of the ER. We performed a similar screen by the electron transfer chain. with Ero1-α, an ER-luminal protein that oxidizes the Trx-like protein A similar reaction mechanism has been proposed for the mam- disulfide isomerase. We found that Ero1-α interacts with most of malian VKOR, with a Trx-like protein in the ER lumen serving as the tested Trx-like proteins, although only poorly with the mem- its redox partner (4, 5, 9). However, conflicting topology models brane-anchored members of the family. Taken together, our results with three or four TM segments have been proposed for human demonstrate that human VKOR employs the same electron VKOR (6, 9, 11); the loop cysteines interacting with the Trx-like transfer pathway as its bacterial homologs and that VKORs gener- protein would thus be on either the cytosolic or luminal side of ally prefer membrane-bound Trx-like redox partners. the ER membrane. In addition, both the cytosolic thioredoxin protein and the luminal PDI protein have been shown to support disulfide bond formation ∣ warfarin ∣ quinone ∣ blood coagulation ∣ the VKOR reaction in vitro (4, 5, 12, 13). The recent crystal struc- electron transfer ture of a bacterial VKOR homolog and multiple sequence align- ments suggest that the mammalian VKORs may also conform to itamin K epoxide reductase (VKOR) is a crucial enzyme in a four-TM model, which would necessitate an ER-luminal redox Vblood coagulation and the target of the most commonly used partner (6, 9). However, there are many potential redox partners, oral anticoagulant warfarin. VKOR catalyzes the reductive as the mammalian ER contains 20 proteins with a Trx-like fold, 12 branch of the vitamin K cycle (for review, see refs. 1 and 2). of which also have at least one CXXC motif (for review, see The cycle begins with the vitamin K-dependent γ-glutamyl ref. 14). The most abundant and best-studied member of this carboxylase modifying select glutamate residues in blood-clotting family is PDI, which is a major catalyst of disulfide bridge forma- factors, a modification required for their activation at sites of tion in newly synthesized secretory and membrane proteins. After tissue injury (for review, see ref. 3). During the carboxylation donating its disulfide bridge to a substrate, PDI is reoxidized by Ero1-α, an ER-luminal protein that uses FAD and molecular reaction, vitamin K hydroquinone is converted to the epoxide – form, and VKOR is needed to regenerate the hydroquinone, thus oxygen in the reaction (15 17). Another member of the ER completing the cycle. The VKOR reaction requires cooperation family of Trx-like proteins, ERp57, has been implicated in the with a redox partner that delivers reducing equivalents. The redox oxidation of glycosylated substrates maintained in the calnexin/ calreticulin folding cycle (18). Conflicting results exist on whether partner remains uncertain, although protein disulfide isomerase α – (PDI), a thioredoxin (Trx)-like protein in the endoplasmic reticu- ERp57 is reoxidized by Ero1- as well (19 21). Although some lum (ER) lumen, has been proposed to serve this role (4, 5). of the other Trx-like ER proteins have been characterized with Homologs of VKOR have been identified in many organisms, respect to redox potential and possible substrates, their biological even species that do not have blood (6–8). These include several functions and physiological oxidants remain unknown. classes of bacteria in which these enzymes are involved in disul- Here, we demonstrate that the human VKOR indeed contains four TM segments, indicating that it interacts with an ER-luminal fide bridge formation of newly synthesized secretory proteins BIOCHEMISTRY (7–10). The reduced cysteines of exported proteins are initially redox partner. To identify this partner, we screened a panel oxidized by a Trx-like protein in the periplasm, leading to the of ER-luminal Trx-like proteins for their ability to form a key reduction of the disulfide bridge in the active site CXXC motif of the Trx-like protein. VKOR then couples the reoxidation of the Author contributions: S.S., B.W., and W.L. performed research; S.S., B.W., W.L., and T.A.R. CXXC motif in the Trx-like protein with reduction of a quinone, analyzed data; and S.S. and T.A.R. wrote the paper. such as vitamin K2 (menaquinone), to the hydroquinone The authors declare no conflict of interest. (Fig. 1A). A recent crystal structure of the VKOR homolog from 1To whom correspondence should be addressed. E-mail: [email protected]. Synechococcus sp., in which the enzyme and a Trx-like domain are This article contains supporting information online at www.pnas.org/lookup/suppl/ contained in a single polypeptide chain, revealed many details doi:10.1073/pnas.1009972107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009972107 PNAS ∣ August 24, 2010 ∣ vol. 107 ∣ no. 34 ∣ 15027–15032 Downloaded by guest on September 25, 2021 weight shift. With wild-type VKOR, little modification was ob- served (Fig. 1B, lane 1). However, extensive modification was observed when the ER membrane was solubilized with Triton X-100 (lane 3 versus lane 4), as expected from the presence of multiple endogenous cysteines located inside the ER membrane and in the ER lumen. By contrast, when a cysteine was introduced at the N terminus (Ser3Cys), it was readily modified by Mal-PEG (lane 5), indicating that the N terminus is located in the cytosol. Similar results were obtained with a cysteine introduced at the C terminus (Gly158Cys), consistent with the expectation that it is also located in the cytosol. Control experiments with the lumi- nal ER protein Grp94 showed that its single cysteine can only be modified when the membrane is solubilized by TX-100 (lanes 3, 7, and 11). These data demonstrate that both termini of VKOR are located in the cytosol, supporting a four-TM model for human VKOR and suggesting that the two loop cysteines engage a redox partner located in the ER lumen (Fig. 1B, Right). To identify potential redox partners of human VKOR among the Trx-like proteins of the ER, we exploited a strategy that is based on the established electron transfer pathway in bacteria (9). The correct interaction partners are expected to form a transient mixed disulfide bridge, in which the N-terminal cysteine in the CXXC motif of the Trx-like protein and the N-terminal loop Fig. 1. Electron transfer pathway and membrane topology of human VKOR. cysteine of VKOR (Cys43) are covalently linked (boxed inter- (A) The scheme shows how electrons flow from reduced cysteines in newly mediate in Fig. 1A). This intermediate can be stabilized by mu- synthesized proteins in the ER lumen through a Trx-like protein and VKOR to tating either the C-terminal loop cysteine of VKOR (Cys51Ala), reduce either vitamin K epoxide (KO) to the quinone (K) or the quinone to the C-terminal cysteine in the CXXC motif of the Trx-like pro- the hydroquinone (KH2). The reaction proceeds through the boxed intermediate in which the N-terminal cysteine of the CXXC motif in the tein, or both. These mutations slow the resolution of the mixed Trx-like protein forms a mixed disulfide bond with Cys43 of VKOR.