The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme

Abstract

The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme. Biochemical Journal, 473(7), pp. 851-858. (doi:10.1042/bj20151223) This is the author's final accepted version.There may be differences between this version and the published version. You are advised to consult the publisher's version if you wish to cite from it.http://eprints.gla.ac.uk/120748/ ABSTRACTThe membrane topology of vitamin K epoxide hydrolase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and it's recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive GFP fused to the N-or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin like transmembrane protein (TMX) to demonstrate ER localisation of the major loop. The topology is identical to the bacterial homologue from Synechococcus sp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase family in recycling VKOR.

Warfarin traps human vitamin K epoxide reductase in an intermediate state during electron transfer

“…The conflicting conclusions of the three- 14,16–18 and four-TM 29,30 models arise from topology analyses that have used modified variants of hVKOR, strongly suggesting that the native conformation can be perturbed by these modifications 31,32 . Here we probed the native topology of hVKOR based on the redox status of cysteines in this oxidoreductase.…”

“…The conflicting three or four-TM models derived from previous studies suggest that the native conformation of hVKOR is sensitive to perturbation, such as truncations of TM domains 14 or fusions with large reporter proteins 16,30 . In general, caution should be taken when analyzing proteins with a flexible structure 31,38,39 , and this example reinforces that caution.…”

“…The MS quantification provides direct evidence for the electron-transfer pathway. Moreover, the analysis of disulfide bonds in hVKOR effectively resolves the debate about its folding topology 14,16–18,29,30 . Furthermore, establishing the catalytic process and architecture of hVKOR provides the basis to understand interactions with warfarin.…”

Intramembrane Thiol Oxidoreductases: Evolutionary Convergence and Structural Controversy

“…This experiment, however, cannot rule out the possibility that the introduced site is buried from glycosylation in the full-length structure, whereas in the short hVKOR variant this region is unstructured and exposed for glycosylation. Indeed, a recent paper showed that, when the glycosylation site was introduced at a different amino acid position, the same region (after TM1) in full-length hVKOR was exposed for glycosylation 38 . Thus, the C-terminus of TM1 is in the ER lumen, as predicted by the four-TM model.…”

From Protein Folding to Blood Coagulation: Menaquinone as a Metabolic Link between Bacteria and Mammals

“…An important caveat to this work is that the protease sensitivity assay was performed after permeabilization with digitonin, a process not thought to affect the topology of membrane proteins. However, a very similar approach using live (i.e., non-permeabilized) cells and a redox-active gfp clearly demonstrated that both the N-and C-termini are located within the cytoplasm [51]. Further experiments showed that the loop region containing Cys43 and Cys51 could be glycosylated by machinery within the ER lumen and that the loop cysteines could form mixed disulfides with luminal proteins, thereby placing this loop region firmly in the ER in accordance with a 4-TM topology.…”