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Vitamin K epoxide reductases (VKORs) are a large family of integral membrane enzymes found from bacteria to humans. Human VKOR, specific target of warfarin, has both the epoxide and quinone reductase activity to maintain the vitamin K cycle. Bacterial VKOR homologs, however, are insensitive to warfarin inhibition and are quinone reductases incapable of epoxide reduction. What affords the epoxide reductase activity in human VKOR remains unknown. Here, we show that a representative bacterial VKOR homolog can be converted to an epoxide reductase that is also inhibitable by warfarin. To generate this new activity, we first substituted several regions surrounding the active site of bacterial VKOR by those from human VKOR based on comparison of their crystal structures. Subsequent systematic substitutions narrowed down to merely eight residues, with the addition of a membrane anchor domain, that are responsible for the epoxide reductase activity. Substitutions corresponding to N80 and Y139 in human VKOR provide strong hydrogen bonding interactions to facilitate the epoxide reduction. The rest of six substitutions increase the size and change the shape of the substrate-binding pocket, and the membrane anchor domain stabilizes this pocket while allowing certain flexibility for optimal binding of the epoxide substrate. Overall, our study reveals the structural features of the epoxide reductase activity carried out by a subset of VKOR family in the membrane environment.

The hematopoietic stem cell (HSC) cycle responds to inflammatory and other proliferative stressors; however, these cells must quickly return to quiescence to avoid exhaustion and maintain their functional integrity. The mechanisms that regulate this return to quiescence are not well understood. Here, we show that tetraspanin CD53 is markedly upregulated in HSCs in response to a variety of inflammatory and proliferative stimuli and that the loss of CD53 is associated with prolonged cycling and reduced HSC function in the context of inflammatory stress. Mechanistically, CD53 promotes the activity of the dimerization partner, RB-like, E2F, and multi-vulva class B (DREAM) transcriptional repressor complex, which downregulates genes associated with cycling and division. Proximity labeling and confocal fluorescence microscopy studies showed that CD53 interacts with DREAM-associated proteins, specifically promoting the interaction between Rbl2/p130 and its phosphatase protein phosphatase 2A (PP2A), effectively stabilizing p130 protein availability for DREAM binding. Together, these data identified a novel mechanism by which stressed HSCs resist cycling.

Tetraspanins are transmembrane signaling and pro-inflammatory proteins. Prior work demonstrates the tetraspanin, CD53/TSPAN25/MOX44 mediates B-cell development, and lymphocyte homing and migration to lymph nodes, and is implicated in various inflammatory diseases including atherosclerosis and microbial infection. However, CD53 is also expressed in highly metabolic tissues, including adipose and liver, yet its function outside of the lymphoid compartment is not defined. Here, we show that CD53 demarcates the nutritional and inflammatory status of hepatocytes. High-fat exposure and inflammatory stimuli induced CD53 in vivo in liver and in isolated primary hepatocytes. In contrast, restricting hepatocyte glucose flux through hepatocyte GLUT8 deletion, or through trehalose treatment blocked CD53 induction in fat- and fructose-exposed contexts. Furthermore, germline CD53 deletion in vivo blocked western diet-induced dyslipidemia and hepatic inflammatory transcriptomic activation. Surprisingly, metabolic protection in CD53 KO mice was more pronounced in the presence of an inciting inflammatory event. CD53 deletion attenuated TNFα-induced and fatty acid + lipopolysaccharide-induced cytokine gene expression and hepatocyte triglyceride accumulation in isolated murine hepatocytes. In vivo, CD53 deletion in non-alcoholic steatohepatitis (NASH)-diet-fed mice blocked peripheral adipose accumulation and adipose inflammation, insulin tolerance, and liver lipid accumulation. We then define a stabilized, trehalase-resistant trehalose polymer that blocks hepatocyte CD53 expression in basal and over-fed contexts. The data suggest that CD53 integrates inflammatory and metabolic signals in response to hepatocyte nutritional status, and that CD53 blockade may be an effective means by which to attenuate pathophysiology in diseases that integrate overnutrition and inflammation, such as NASH and type 2 diabetes mellitus.

Intramembrane enzymes are often difficult for biochemical characterization. Human vitamin K epoxide reductase (VKOR) is the target of warfarin. However, this intramembrane enzyme becomes insensitive to warfarin inhibition in vitro, preventing the characterization of inhibition kinetics for decades. Here we employ structural biology methods to identify stable VKOR and VKOR-like proteins and purify them to near homogeneity. We find that the key to maintain their warfarin sensitivity is to stabilize their native protein conformation in vitro. Reduced glutathione drastically increases the warfarin sensitivity of a VKOR-like protein from Takifugu rubripes, presumably through maintaining a disulfide-bonded conformation. Effective inhibition of human VKOR-like requires also the use of LMNG, a mild detergent developed for crystallography to increase membrane protein stability. Human VKOR needs to be preserved in ER-enriched microsomes to exhibit warfarin sensitivity, whereas human VKOR purified in LMNG is stable only with pre-bound warfarin. Under these optimal conditions, warfarin inhibits with tight-binding kinetics. Overall, our studies show that structural biology methods are ideal for stabilizing intramembrane enzymes. Optimizing toward their inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo-electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS-based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

Maintenance of quiescence is necessary for optimal hematopoietic stem cell (HSC) function. The absence of fine-tuned cycling regulation of HSCs can result in impaired hematopoiesis, bone marrow (BM) failure, or malignant transformation. While various factors, including inflammatory cytokines and chemotherapy, have been identified to induce HSC cycling, the mechanisms regulating HSC return to quiescence are unclear. Herein, we profiled HSCs in response to a variety of inflammatory stressors (cytokines, toll-like receptor agonists, mobilizing agents, and chemotherapeutics) and found markedly upregulated expression of CD53 in both mouse and human HSCs, in some cases by as much as 70-fold higher expression over vehicle-treated controls. CD53 is a tetraspanin, a type of transmembrane protein involved in plasma membrane organization and regulation of processes such as cellular migration, adhesion, and signaling. CD53 has been shown to be asymmetrically segregated in HSCs, with CD53-enriched HSCs believed to be more stem-like; however, there is no current proposed mechanism to explain the association between CD53 and stem cell quality. To understand the role of CD53 in HSC quality, we generated a CD53 knockout mouse, and profiled HSC phenotype and function. Under homeostatic conditions, Cd53 -/- HSC number and frequency are normal as compared to wild type (WT) mice. However, we found Cd53 -/-HSCs to have significantly impaired function, particularly in response to inflammatory stimuli. Cd53 -/- BM failed to engraft as well as WT BM (45% chimerism vs 63%, p Transcriptomic sequencing revealed significant upregulation of genes associated with cell cycling and division in G-CSF-treated Cd53 -/- HSCs compared to WT controls. Notably, these differentially expressed genes are targets of the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) complex, a newly described transcriptional regulator that represses cell cycling-associated genes, suggesting that CD53 promotes DREAM-mediated quiescence in stressed HSCs. We performed CUT&Tag profiling of Rbl2/p130, a DREAM complex subunit, in HSCs and found that loss of CD53 was associated with decreased complex binding (consistent with enhanced transcription of cell cycle genes). Proximity labeling studies demonstrated that CD53 interacts with cell cycle machinery proteins, including Rbl2/p130, and Western blots of sorted HSCs from Cd53 -/- and WT mice treated with G-CSF showed decreased expression of DREAM complex components (Rbl1/p107 and Rbl2/p130) in the absence of CD53. Finally, enforced activation of DREAM and HSC quiescence using the CDK4/6 inhibitor palbociclib rescued the Cd53 -/-HSC repopulating defect. Together, these data derive a novel mechanism whereby CD53 regulates HSC quiescence through regulating DREAM complex binding during stress-induced cycling. Disclosures No relevant conflicts of interest to declare.

During oxidative protein folding, disulfide bond formation is catalyzed by thiol oxidoreductases. Through dedicated relay pathways, the disulfide is generated in donor enzymes, passed to carrier enzymes, and subsequently delivered to target proteins. The eukaryotic disulfide donors are flavoenzymes, Ero1 in the endoplasmic reticulum and Erv1 in mitochondria. In prokaryotes, disulfide generation is coupled to quinone reduction, catalyzed by intramembrane donor enzymes, DsbB and VKOR. To catalyze de novo disulfide formation, these different disulfide donors show striking structural convergence at several levels. They share a four-helix bundle core structure at their active site, which contains a CXXC motif at a helical end. They have also evolved a flexible loop with shuttle cysteines to transfer electrons to the active site and relay the disulfide bond to the carrier enzymes. Studies of the prokaryotic VKOR, however, have stirred debate about whether the human homologue adopts the same topology with four transmembrane helices and uses the same electron-transfer mechanism. The controversies have recently been resolved by investigating the human VKOR structure and catalytic process in living cells with a mass spectrometry-based approach. Structural convergence between human VKOR and the disulfide donors is found to underlie cofactor reduction, disulfide generation, and electron transfer.

Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs.

The UbiA superfamily of intramembrane prenyltransferases catalyzes a key biosynthetic step in the production of ubiquinones, menaquinones, plastoquinones, hemes, chlorophylls, vitamin E, and structural lipids. These lipophilic compounds serve as electron and proton carriers for cellular respiration and photosynthesis, as antioxidants to reduce cell damage, and as structural components of microbial cell walls and membranes. This article reviews the biological functions and enzymatic activities of representative members of the superfamily, focusing on the remarkable recent research progress revealing that the UbiA superfamily is centrally implicated in several important physiological processes and human diseases. Because prenyltransferases in this superfamily have distinctive substrate preferences, two recent crystal structures are compared to illuminate the general mechanism for substrate recognition.

The x-ray structure of the monotopic membrane protein (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida reveals an inherent flexibility of its membrane binding segment that might be important for its biological activity. The surface of MDH exhibits a concentration of the positive charges on one side and the negative charges on the other side. The putative membrane binding surface of MDH has a concentric circular ridge, formed by positively charged residues, which projects away from the protein surface by ∼4 A; this is an unique structural feature and not observed in other monotopic membrane proteins to our knowledge. There are three α-helixes in the membrane binding region. Based on the structure of MDH, it is possible to propose that the interaction of MDH with the membrane is stabilized by coplanar electrostatic interactions, between the positively charged concentric circular ridge and the negatively charged head-groups of the phospholipid bilayer, along with three α-helixes that provide additional stability by inserting into the membrane. The structure reveals the possible orientation of these helixes along with possible roles for the individual residues which form those helixes. These α-helixes may play a role in the enzyme's mobility. A detergent molecule, N-Dodecyl-β-maltoside, is inserted between the membrane binding region and rest of the molecule and may provide structural stability to intra-protein regions by forming hydrogen bonds and close contacts. From the average B-factor of the MDH structure, it is likely that MDH is highly mobile, which might be essential for its interaction in membrane and non-membrane environments, as its substrate (S)-mandelate, is from the cytoplasm, while its electron acceptor is a component of the membrane electron transport chain.

Mass spectrometry-based footprinting is an emerging approach for studying protein structure. Because integral membrane proteins are difficult targets for conventional structural biology, we recently developed a mass spectrometry (MS) footprinting method to probe membrane protein-drug interactions in live cells. This method can detect structural differences between apo and drug-bound states of membrane proteins, with the changes inferred from MS quantification of the cysteine modification pattern, generated by residue-specific chemical labeling. Here, we describe the experimental design, interpretation, advantages, and limitations of using cysteine footprinting by taking as an example the interaction of warfarin with vitamin K epoxide reductase, a human membrane protein. Compared with other structural methods, footprinting of proteins in live cells produces structural information for the near native state. Knowledge of cellular conformational states is a necessary complement to the high-resolution structures obtained from purified proteins in vitro. Thus, the MS footprinting method is broadly applicable in membrane protein biology. Future directions include probing flexible motions of membrane proteins and their interaction interface in live cells, which are often beyond the reach of conventional structural methods.

The UbiA superfamily is a group of intramembrane prenyltransferases that generate lipophilic compounds essential in biological membranes. These compounds, which include various quinones, hemes, chlorophylls, and vitamin E, participate in electron transport and function as antioxidants, as well as acting as structural lipids of microbial cell walls and membranes. Prenyltransferases producing these compounds are involved in important physiological processes and human diseases. These UbiA superfamily members differ significantly in their enzymatic activities and substrate selectivities. This chapter describes examples of methods that can be used to group these intramembrane enzymes, analyze their activity, and screen and crystallize homolog proteins for structure determination. Recent structures of two archaeal homologs are compared with structures of soluble prenyltransferases to show distinct mechanisms used by the UbiA superfamily to control enzymatic activity in membranes.

Realgar is a type of mineral drug containing arsenic. The nervous system toxicity of realgar has received extensive attention. However, the underlying mechanisms of realgar-induced neurotoxicity have not been clearly elucidated. To explore the mechanisms that contribute to realgar-induced neurotoxicity, weanling rats were exposed to realgar (0, 0.3, 0.9, 2.7 g/kg) for 6 weeks, and cognitive ability was tested using the Morris water maze (MWM) test and object recognition task (ORT). The levels of arsenic in the blood and hippocampus were monitored. The ultrastructures of hippocampal neurons were observed. The levels of glutamate (Glu) and glutamine (Gln) in the hippocampus and hippocampal CA1 region; the activities of glutamine synthetase (GS) and phosphate-activated glutaminase (PAG); the mRNA and protein expression of glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST), and N-methyl-D-aspartate (NMDA) receptors; and the level of intracellular Ca(2+) were also investigated. The results indicate that the rats developed deficiencies in cognitive ability after a 6-week exposure to realgar. The arsenic contained in realgar and the arsenic metabolites passed through the blood-brain barrier (BBB) and accumulated in the hippocampus, which resulted in the excessive accumulation of Glu in the extracellular space. The excessive accumulation of Glu in the extracellular space induced excitotoxicity, which was shown by enhanced GS and PAG activities, inhibition of GLT-1 mRNA and protein expression, alterations in NMDA receptor mRNA and protein expression, disturbance of intracellular Ca(2+) homeostasis, and ultrastructural changes in hippocampal neurons. In conclusion, the findings from our study indicate that exposure to realgar induces excitotoxicity and that the mechanism by which this occurs may be associated with disturbances in Glu metabolism and transportation and alterations in NMDA receptor expression.

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane-embedded enzyme difficult to characterize with structural and biochemical studies. Here we achieve a long-standing goal of obtaining crystal structures of human VKOR with warfarin, which represents coumarin-based VKAs; with phenindione, which represents indandione-based VKAs; with superwarfarins, the most commonly used rodenticides; and with vitamin K epoxide in a reaction intermediate state. We have also solved structures of a VKOR-like homolog with warfarin, with vitamin K substrates, and without ligand. These structures show that human VKOR adopts an overall fold with four transmembrane helices (TM) and a large ER-luminal region. VKAs are bound at the active site of HsVKOR, which is formed by the surrounding four-TM bundle and a cap domain on top. The cap domain is stabilized by a linked anchor domain that interacts with the membrane surface. VKOR binds specifically to VKAs through hydrogen bonding to their diketone groups. Mutating VKOR residues recognizing the diketones render strong warfarin resistance. Except the hydrogen bonds, the binding pocket is largely hydrophobic. This pocket is incompatible with warfarin metabolite, explaining the inactivation of warfarin through CYP2C9 metabolism; CYP2C9 and VKOR genotypes can explain 30-50% of the patient variability in warfarin dose. In addition, the high potency of superwarfarins is due to the interaction of their side group with a tunnel where the isoprenyl chain of vitamin K is bound. For VKOR catalysis, the same residues affording the VKA-binding specificity also facilitate substrate reduction Initiation of the catalysis requires a reactive cysteine to form a substrate adduct. Interactions from this stably bound adduct induces a closed conformation, thereby triggering electron transfer to reduce the substrate. Importantly, the open to closed conformational change during catalysis similar to that induced by the binding of VKAs. Taken together, VKAs achieve inhibition through mimicking key interactions and conformational changes required for VKOR catalytic cycle. Understanding of these mechanisms will enable improved strategy to regulate warfarin dose and have a broad impact on thromboembolic diseases and bone disorders. Disclosures No relevant conflicts of interest to declare.

Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the γ-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-α, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-α interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.

Catalysis in the Membrane Enzymes in the UbiA superfamily of integral membrane proteins synthesize lipid-soluble aromatics such as ubiquinones and chlorophylls that function in energy storage and energy transfer in mitochondrial and chloroplast membranes. Cheng and Li (p. 878 ) report structures of an archaeal UbiA protein in both apo and substrate-bound states. The structures show a large active site with a lateral portal that is likely to give access to the long-chain isoprenoid substrates. The findings suggest a mechanism for substrate recognition and catalysis and can explain disease-related mutants in eukaryotic homologs.

The intramembrane vitamin K epoxide reductase (VKOR) supports blood coagulation in humans and is the target of the anticoagulant warfarin. VKOR and its homologues generate disulphide bonds in organisms ranging from bacteria to humans. Here, to better understand the mechanism of VKOR catalysis, we report two crystal structures of a bacterial VKOR captured in different reaction states. These structures reveal a short helix at the hydrophobic active site of VKOR that alters between wound and unwound conformations. Motions of this 'horizontal helix' promote electron transfer by regulating the positions of two cysteines in an adjacent loop. Winding of the helix separates these 'loop cysteines' to prevent backward electron flow. Despite these motions, hydrophobicity at the active site is maintained to facilitate VKOR catalysis. Biochemical experiments suggest that several warfarin-resistant mutations act by changing the conformation of the horizontal helix. Taken together, these studies provide a comprehensive understanding of VKOR function.

Realgar is a traditional Chinese medicine, which has been used for thousands of years and are claimed to have therapeutic effects. The toxicity from realgar or realgar-containing traditional medicines has raised public concern. However, the neurotoxicity induced by realgar is less reported. Amino acid neurotransmitters are closely linked to the vulnerability of the immature brain to neuronal injury. The investigation of amino acid neurotransmitters is important to understand the evolution of developmental brain damage. An improved HPLC–UV method was developed and applied to analyzing amino acid neurotransmitters of aspartate, glutamate, glutamine, homocysteine, serine, glycine, γ-aminobutyric acid and taurine in brain tissues of immature rats after the treatment of realgar. Significant changes of these amino acid neurotransmitters were observed in realgar treated groups. Negative correlations were found between the levels of some amino acids and the contents of arsenic in brain tissues. The result indicates that the neurotoxicity induced by realgar is associated with its effects on amino acid neurotransmitters.

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain γ-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. Mammalian vitamin K epoxide reductase (VKOR) catalyses the generation of vitamin K hydroquinone, a decisive step in the vitamin K cycle that is required to sustain blood coagulation. The X-ray crystal structure of a bacterial homologue of VKOR has now been determined. It shows VKOR in a complex with its redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. This points to a possible mechanism by which VKOR uses electrons from newly synthesized proteins to reduce the quinone. This work may help explain how mutations to VKOR cause resistance to warfarin, the ubiquitous anticoagulant that acts by inhibiting VKOR. The γ-carboxylation of many blood coagulation factors relies on the generation of vitamin K hydroquinone by the enzyme vitamin K epoxide reductase (VKOR), of which the anticoagulant warfarin is an inhibitor. Here, the X-ray crystal structure of a bacterial homologue of VKOR is presented; the results have implications for the mechanism of action of mammalian VKOR and explain how mutations can cause warfarin resistance.

Many Gram-negative bacteria have two cytoplasmic thioredoxins, thioredoxin-1 and -2, encoded by the trxA and trxC genes, respectively. Both thioredoxins have the highly conserved WCGPC motif and function as disulfide-bond reductases. However, thioredoxin-2 has unique features: it has an N-terminal motif that binds a zinc ion, and its transcription is under the control of OxyR, which allows it to be up-regulated under oxidative stress. Here, we report the crystal structure of thioredoxin-2 from Rhodobacter capsulatus. The C-terminal region of thioredoxin-2 forms a canonical thioredoxin fold with a central beta-sheet consisting of five strands and four flanking alpha-helices on either side. The N-terminal zinc finger is composed of four short beta-strands (S1-S4) connected by three short loops (L1-L3). The four cysteines are at loops L1 and L3 and form a tetragonal binding site for a zinc ion. The zinc finger is close to the first beta-strand and first alpha-helix of the thioredoxin fold. Nevertheless, the zinc finger may not directly affect the oxidoreductase activity of thioredoxin-2 because the zinc finger is not near the active site of a protomer and because thioredoxin-2 is a monomer in solution. On the basis of structural similarity to the zinc fingers in Npl4 and Vps36, we propose that the N-terminal zinc finger of thioredoxin-2 mediates protein-protein interactions, possibly with its substrates or chaperones.

Warfarin targets vitamin K epoxide reductase (VKOR) to interfere with blood coagulation in humans. Warfarin is the most widely used oral anticoagulant, but its inhibition mechanism remains largely unknown. Here we use quantitative mass spectrometry to show that warfarin changes the intracellular redox state of human VKOR. Warfarin induces this redox shift at a physiological dosage that correlates well with warfarin-inhibited VKOR activity. The warfarin-induced redox change is prevented by the mutation of two critical cysteines, suggesting that they form a disulfide bond essential for warfarin binding. We also mapped warfarin binding through its interaction with warfarin resistant mutations located at different structural regions of human VKOR. Modeling of these interactions suggests how warfarin binds to a VKOR conformation that is stabilized by the essential disulfide bond. Thus, after sixty years of clinical use of warfarin, we now have a good mechanistic understanding that this drug blocks human VKOR at a specific conformational and redox state. Disclosures No relevant conflicts of interest to declare.

The stability of porcine brain inositol monophosphatase in the presence of increasing concentrations of urea was investigated at pH 7.5. Exposure of the enzyme to 8 M urea brings about the dissociation of the dimeric species of 58 kDa into monomeric forms as revealed by gel filtration chromatography. Unfolding of the protein by 8 M urea results in a decrease of the ellipticity at 220 nm (20%) together with a perturbation of the near-UV circular dichroism spectrum. Urea-treated inositol monophosphatase binds Co2+ ions with a dissociation constant of 3.3 microM. The enzyme is catalytically competent when assayed with 4-nitrophenyl-phosphate in the presence of the activating ion Co2+ at pH 7.5 in 8 M urea. The apparent activation constant for Co2+ is 2.5 mM. It is postulated that partially folded conformations of monomeric species preserve their catalytic function because the affinity of Co2+ ions for the metal coordination center of the protein is not perturbed by exposure to 8 M urea.

Vitamin K epoxide reductase (VKOR) is an intramembrane enzyme required for blood coagulation and is the target of the anticoagulant warfarin. The reduction of vitamin K epoxide is coupled with disulfide-bond formation at the active site of VKOR. To regenerate the active site, VKOR is reduced by protein partners that transfer electrons to VKOR. Here we report two crystal structures of a bacterial VKOR homolog with its reducing partner captured in different conformational states. These structures reveal a short helix at the hydrophobic active site of VKOR that undergoes stretching and compressing motions. Motions of this “horizontal helix” promote electron transfer to the VKOR active site by regulating the positions of two cysteines in an adjacent loop. Compression of the helix also separates the “loop cysteines” to prevent backward electron transfer. During these motions, hydrophobicity at the active site is maintained to facilitate vitamin K reduction. Biochemical experiments support the structural model and suggest that several warfarin-resistant mutations act by changing the conformation of the horizontal helix. Taken together, these structures provide a comprehensive understanding of electron transfer and vitamin K reduction catalyzed by VKOR. Disclosures: No relevant conflicts of interest to declare.