Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain γ-carboxylation of many blood coagulation factors. Here, we report the 3.6Å crystal structure of a bacterial homolog 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 newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulfide 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.

Key Points Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders. The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.

Autocatalytic processing of the Hedgehog ligand from its precursor protein relies on protein disulfide isomerase and ER-associated degradation.

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.disulfide bond formation | warfarin | quinone | blood coagulation | electron transfer

Key Points• A gain-of-function variant in DIAPH1 causes macrothrombocytopenia and hearing loss and extends the spectrum of DIAPH1-related disease.• Our findings of altered megakaryopoiesis and platelet cytoskeletal regulation highlight a critical role for DIAPH1 in platelet formation.Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease andprovides new insight into the autoregulation of DIAPH1 activity.

Proteins are translocated across membranes through a channel that is formed by the prokaryotic SecY or eukaryotic Sec61 complex. The crystal structure of the SecY channel from M. jannaschii revealed a plug domain that appears to seal the channel in its closed state. However, the role of the plug remains unclear, particularly because plug deletion mutants in S. cerevisiae are functional. Here, we demonstrate that plug deletion mutants in E. coli SecY are also functional and even efficiently translocate proteins with defective or missing signal sequences. The crystal structures of equivalent plug deletions in SecY of M. jannaschii show that, although the overall structures are maintained, new plugs are formed. These lack many interactions that normally stabilize the closed channel, explaining why the channels can open for proteins with signal-sequence mutations. Our data show that the plug domain is required to maintain a closed state of the channel and suggest a mechanism for channel gating.

BackgroundHeritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases.MethodsWe report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes.ResultsWe show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician.ConclusionsThese findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0151-5) contains supplementary material, which is available to authorized users.

The channel formed by the SecY complex must maintain the membrane barrier for ions and other small molecules during the translocation of membrane or secretory proteins. We have tested the permeability of the channel by using planar bilayers containing reconstituted purified E. coli SecY complex. Wild-type SecY complex did not show any conductance for ions or water. Deletion of the "plug," a short helix normally located in the center of the SecY complex, or modification of a cysteine introduced into the plug resulted in transient channel openings; a similar effect was seen with a mutation in the pore ring, a constriction in the center of the channel. Permanent channel opening occurred when the plug was moved out of the way by disulfide-bridge formation. These data show that the resting channel on its own forms a barrier for small molecules, with both the pore ring and the plug required for the seal; channel opening requires movement of the plug.