Jian Ke Tie

Structure and Function of Vitamin K Epoxide Reductase

Vitamin K epoxide reductase (VKOR) is an integral membrane protein that catalyzes the reduction of vitamin K 2,3-epoxide and vitamin K to vitamin K hydroquinone, a cofactor required for the gamma-glutamyl carboxylation reaction. VKOR is highly sensitive to inhibition by warfarin, the most commonly prescribed oral anticoagulant. Warfarin inhibition of VKOR decreases the concentration of reduced vitamin K, which reduces the rate of vitamin K-dependent carboxylation and leads to under-carboxylated, inactive vitamin K-dependent proteins. It is proposed that an active site disulfide needs to be reduced for the enzyme to be active. VKOR uses two sulfhydryl groups for the catalytic reaction and these two sulfhydryl groups are oxidized back to a disulfide bond during each catalytic cycle. The recent identification of the gene encoding VKOR allows us to study its structure and function relationship at the molecular level. The membrane topology model shows that VKOR spans the endoplasmic reticulum membrane three times with its amino-terminus residing in the lumen and the carboxyl-terminus residing in the cytoplasm. Both the active site (cysteines 132 and 135) and the proposed warfarin binding site (tyrosine 139) reside in the third transmembrane helix. VKOR is made at high levels in insect cells and is relatively easily purified. This should allow the determination of its three-dimensional structure. A detailed mechanism has been published and the purified enzyme should allow the testing of this mechanism. A major unanswered question is the physiological reductant of VKOR.

Functional study of the vitamin K cycle in mammalian cells

We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH₂) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH₂, and (4) the primary function of VKOR is the reduction of KO to vitamin K.

The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

The vitamin K-dependent γ-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997–1998) noticed that residues 495–513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Prices hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K d values of these mutant enzymes for human factor IX propeptide varied from 0.5- to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparentK d values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide andV max. Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding.

Human Vitamin K Epoxide Reductase and Its Bacterial Homologue Have Different Membrane Topologies and Reaction Mechanisms

Background: The membrane topology and the role of certain cysteines in human vitamin K epoxide reductase (VKOR) are disputed. Results: VKOR has three transmembrane domains, and only the active site cysteines are required for function. Conclusion: Despite similarities, VKOR and its bacterial homologues (VKORHs) have different topologies and reaction mechanisms. Significance: The VKOR does not employ the intramolecular electron transfer pathway proposed for VKORH. 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.

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

Background: The structure and the physiological function(s) of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are unknown. Results: VKORC1L1 has four transmembrane domains and employs an intra-molecular electron transfer pathway for active site regeneration. Conclusion: The different structure and reaction mechanism of VKORC1L1, as compared with VKORC1, suggest that VKORC1L1 has different physiological function(s). Significance: Four conserved cysteines in VKORC1L1 function in concert for active site reduction. Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1s active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1s overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1s active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Mycobacterium tuberculosis Vitamin K Epoxide Reductase Homologue Supports Vitamin K–Dependent Carboxylation in Mammalian Cells

AIMS Vitamin K epoxide reductase complex, subunit 1 (VKORC1) is a critical participant in the production of active forms of reduced vitamin K and is required for modification of vitamin K-dependent proteins. Homologues of VKORC1 (VKORH) exist throughout evolution, but in bacteria they appear to function in oxidative protein folding as well as quinone reduction. In the current study we explore two questions: Do VKORHs function in the mammalian vitamin K cycle? Is the pair of loop cysteines-C43 and C51 in human VKORC1-conserved in all VKORC1s, essential for the activity of vitamin K epoxide reduction? RESULTS We used our recently developed cell-based assay to compare the function of VKORHs to that of human VKORC1 in mammalian cells. We identified for the first time a VKORH (from Mycobacterium tuberculosis [Mt-VKORH]) that can function in the mammalian vitamin K cycle with vitamin K epoxide or vitamin K as substrate. Consistent with our previous in vitro results, the loop cysteines of human VKORC1 are not essential for its activity in vivo. Moreover, the corresponding loop cysteines of Mt-VKORH (C57 and C65), which are essential for its activity in disulfide bond formation during protein folding in Escherichia coli, are not required in the mammalian vitamin K cycle. INNOVATION AND CONCLUSIONS Our results indicate that VKORC1 in eukaryotes and Mt-VKORH in bacteria, that is, in their respective native environments, employ apparently different mechanisms for electron transfer. However, when Mt-VKORH is in the mammalian cell system, it employs a mechanism similar to that of VKORC1.

Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase †

We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylases transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.

Characterization of vitamin K–dependent carboxylase mutations that cause bleeding and nonbleeding disorders

Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix γ-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patients GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo.

Vitamin K epoxide reductase and its paralogous enzyme have different structures and functions

Vitamin K epoxide reductase (VKOR) is an essential enzyme for vitamin K-dependent carboxylation, while the physiological function of its paralogous enzyme VKOR-like (VKORL) is yet unknown. Although these two enzymes share approximately 50% protein sequence homology, the membrane topology of VKOR is still in debate. Here, we explored the differences in the membrane topology and disulfide-linked oligomerization of these two enzymes. Results from mutating the critical amino acid residues in the disputed transmembrane (TM) regions revealed that the second TM domain in the proposed 4-TM model of VKOR does not function as an authentic TM helix; supporting VKOR is a 3-TM protein, which is different from VKORL. Additionally, altering the loop sequence between the two conserved cysteine residues of VKORL affects its activity, supporting the notion that the conserved loop cysteines of VKORL are involved in its active site regeneration. However, a similar mutation in VKOR does not affect its enzymatic activity. Finally, our results show that although both VKOR and VKORL form disulfide-linked oligomers, the cysteine residues involved in the oligomerization appear to be different. Overall, the structural and functional differences between VKOR and VKORL shown here indicate that VKORL might have a different physiological function other than recycling vitamin K.

Molecular basis of the first reported clinical case of congenital combined deficiency of coagulation factors

To the editor: Congenital combined vitamin K–dependent coagulation factors deficiency (VKCFD) is a rare autosomal recessive bleeding disorder.[1][1] Patients with VKCFD have decreased activity in multiple vitamin K–dependent coagulation factors due to genetic mutations that limit the ability of