Structure of a bacterial homologue of vitamin K epoxide reductase

Abstract

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain gamma-carboxylation of many blood coagulation factors. Here, we report the 3.6 A crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

Figure 1. Architecture of Synechococcus VKOR in complex with its redox partner

a, Overall structure of the protein, consisting of VKOR and the thioredoxin (Trx)-like domain. TMs 1-4 that are homologous to the mammalian VKOR are colored in pink. The loop between TMs 1 and 2, containing the 1/2-segment and 1/2-helix, is shown in red. Ubiquinone (UQ) is shown in stick presentation. Sulfur atoms in cysteines are indicated in green. The dotted lines indicate the membrane boundaries. b, View of VKOR from the periplasm. The Trx-like domain was removed for clarity.

Figure 2. The active site of VKOR

a, The active site of VKOR, including the cysteines of the CXXC motif in TM4 and the ubiquinone molecule (UQ). The experimental electron density map is contoured at 1σ (blue) and 3.5σ (red). Note the strong electron density between Cys130 and Cys133, indicative of a disulfide bridge (in green), as well as between Cys133 and the C1 position of the quinone. TM3 and the 1/2-helix are shown without electron density for orientation. b, Surface representation of the binding pocket for the quinone (in red), viewed from the side. TM5 was removed for clarity. Note the cleft between TMs 2 and 3, blocked only by the side chains shown in purple, which could provide a lateral exit for the quinone into lipid. The lower part of the membrane is not shown.

Figure 3. Mutations causing warfarin resistance in mammalian VKOR

a, Membrane topology of VKORs. TMs 1-4 form the essential four-helix bundle found in all VKORs. Dotted lines indicate TM5 and the linker to the Trx-like domain found in Synechococcus sp. The conserved cysteines and serine/threonine are shown as red ovals. Warfarin resistance mutations in mammalian VKOR located close to the bound quinone or elsewhere are indicated as blue or green ovals, respectively. b, Mutations indicated in blue in a are mapped into the Synechococcus structure, based on sequence alignment (Fig. S2). Some TM segments are removed for clarity. Labels refer to the residue in Synechococcus VKOR, followed by the residue and its position in human VKOR. The positions V29, R33, and D36 are in a helical extension of TM1, but their location is somewhat uncertain because of alignment ambiguities.

Figure 4. Electron transfer pathway

The scheme shows the flow of electrons from a newly synthesized protein (substrate) in the periplasm of bacteria or the ER lumen of eukaryotes to a quinone (UQ). The different states are indicated by Roman numerals. The boxed state corresponds to an intermediate state represented by the crystal structure. A similar intermediate was detected in vivo .

Figure 5. In vitro reconstitution of vitamin K-dependent oxidative folding

a, Purified Synechococcus protein, containing the VKOR and Trx-like domains, was incubated with reduced, denatured RNAse A and vitamin K1 (Vit K1) (lanes 9, 10). The reaction was quenched with AMS, which modifies free sulfhydryl groups. Samples were analyzed by SDS-PAGE and Coomassie staining. Controls were performed without Vit K1 (in ethanol, EtOH) or VKOR, as indicated. RNAse A was also oxidized with glutathione (GSH/GSSG) (lanes 3,4). b, As in a, but with VKOR serially diluted. c, As in a, but with wild type (WT) or mutants in cysteines in the predicted electron transfer pathway (Fig. 4). d, As in c, except reduction of Vit K1 was followed in a fluorometer, using reduced RNAse or dithiothreitol (DTT) as reductant. Error bars represent standard error of mean instrumental fluorescence variation.

Figure 6. Comparison of VKOR with DsbB

a, Superposition of the four-helix bundles of VKOR (pink) and DsbB (yellow) , based on the helices containing the CXXC motifs. The left two panels show views from the side and periplasm. The right panel shows a close-up view of the active sites. b, Overall architecture of VKOR with its Trx-like redox partner (left panel) and of DsbB with its redox partner DsbA . The dotted line in the right panel indicates a segment disordered in the X-ray structure. c, Electron transfer pathways for VKOR and DsbB. Note that for VKOR the loop cysteine cannot access the active site CXXC motif unless the 1/2-helix is displaced. By contrast, in DsbB the 3/4-helix is peripheral and electron transfer can occur without major conformational change; the loop cysteines could not interact if the helix were positioned as in VKOR.