Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms

Abstract

Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.

FIGURE 1.

Proposed membrane topology models of human VKOR. A, three-TMD topology model of human VKOR based on in vitro translation/co-translocation (21). The N terminus of VKOR is located in ER lumen and the C terminus in the cytoplasm. Conserved active site and loop cysteines are indicated by gradient filling of the circle, and they are located on the opposite side of ER membrane. The positively charged residues flanking TMD1 are shaded in gray. B, proposed four-TMD topology model of human VKOR based on the crystal structure of a bacterial VKOR homologue (26). In this model, both the N and C terminus are located in the cytoplasm. Conserved active site cysteines and the loop cysteines are located in the same side of the ER membrane.

FIGURE 2.

Localization of the N and C termini of VKOR in HEK293 cells by fluorescence protease protection assay. A, FPP assay of GFP fusions of human VKOR. GFP-tagged VKOR fusions were transiently expressed in HEK293 cells. Forty eight hours post-transfection, cells were selectively permeabilized with 0.1 mm digitonin. Then trypsin was added, and images were collected at the indicated time intervals. B, schematic representation of the single TMD model proteins (ASGPR and CYB5) and their GFP fusions. The solid bars indicate the TMD; the gray bars indicate the lumenal carbohydrate recognition domain (CMD) of ASGPR or the cytoplasmic heme-binding domain (HBD) of CYB5. Dark gray bars indicate the (GGSGG)₆ linker between GFP and the model proteins. C, FPP assay of GFP fusion of the model proteins as described above.

FIGURE 3.

In vivo cell-based activity assay of human VKOR and its GFP fusions used in FPP assay. Human VKOR and its GFP fusions were transiently expressed in FIXgla-PC/HEK293 cells. Transfected cells were cultured in complete medium containing 5 μm KO and 4 μm warfarin for 48 h. The concentration of carboxylated FIXgla-PC in the cell culture medium was measured by ELISA and normalized by the luciferase activity as described under “Experimental Procedures.” Data are presented as mean ± S.D. (n = 3).

FIGURE 4.

Selective modification of the endogenous cysteine residues of human VKOR and its C43A/C51A cysteine mutant by mPEG-MAL-5000. A, C-terminal HPC4-tagged VKOR or its C43A/C51A mutant was transiently expressed in HEK293 cells. Forty eight hours post-transfection, cells were harvested and permeabilized with digitonin or Triton X-100 before mPEG-MAL modification. Whole cell lysate were applied to SDS-PAGE and transferred to PVDF membrane; HPC4-tagged VKOR was analyzed by Western blot. B, hydrophobicity profile of VKOR and the location of the endogenous cysteine residues. Transmembrane regions determined by in vitro translation/co-translocation were marked as TMD1 to TMD3. The putative transmembrane region was marked as pTMD in gray.

FIGURE 5.

FPP topology assay and in vivo cell-based activity assay of charged residue-mutated VKOR. A, FPP topology assay of charged residue-mutated VKOR, VKOR-CM, as described in the legend to Fig. 2. B, time course of relative fluorescence intensity in FPP assay of GFP fusions of VKOR-CM compared with the N-terminal GFP fusion of wild-type VKOR. C, cell-based activity assay of charged residue mutated VKOR and its GFP fusions as described in the legend for Fig. 3.

FIGURE 6.

In vivo cell-based activity assay of the cysteine mutants of human VKOR and VKOR-CM. Cysteine mutants of VKOR or VKOR-CM were transiently expressed in FIXgla-PC/HEK293 cells, and the enzymatic activity was determined as described in the legend to Fig. 3.

FIGURE 7.

In vivo cell-based activity assay of human VKOR and its V54P mutant. A, sequence alignment of VKORs from different species per Ref. . Sequences were truncated between TMD1 and TMD2 as in the four-TMD topology model. Conserved loop cysteines and conserved serine are highlighted in gray. The conserved valine in the middle of the 1/2-helix is boxed. The numbers at the top refer to the position of amino acid residues in the human VKOR sequence. B, human VKOR and its V54P mutant were transiently expressed in FIXgla-PC/HEK293 cells, and the enzymatic activity was determined as described in the legend to Fig. 3.

FIGURE 8.

Charge distribution of human VKOR and Syn-VKORH in the proposed topology models. A, net charge distribution in the three-TMD model of human VKOR and the four-TMD model of the VKOR domain of Syn-VKORH. Net charges of the loops and the termini are calculated according to the rules by Hartmann et al. (39) and are indicated inside the circle. Arginine, lysine, and the N-terminal amino group were given a value of +1, histidine +0.5, and aspartate and glutamate −1 as described previously. B, net charge distribution in the proposed four-TMD model of human VKOR. The putative TMD that functions as an authentic TMD in the four-TMD variant is shown in light gray with dotted line.