Membrane topology and mutational analysis of Mycobacterium tuberculosis VKOR, a protein involved in disulfide bond formation and a homologue of human vitamin K epoxide reductase

Abstract

We have presented evidence that a homologue of vertebrate membrane protein vitamin K epoxide reductase (VKOR) is an important component of the protein disulfide bond-forming pathway in many bacteria. Bacterial VKOR appears to take the place of the nonhomologous DsbB found in Escherichia coli. We also determined the structure of a VKOR from a Cyanobacterium and showed that two or four conserved cysteines are required, according to different reductants for activity in an in vitro assay. Here we present evidence for the topologic arrangement in the cytoplasmic membrane of the VKOR from Mycobacterium tuberculosis (Mtb). The results show that Mtb VKOR is a membrane protein that spans the membrane 5 times with its N-terminus in the cytoplasm, C-terminus in the periplasm, and all four cysteines facing the periplasm. The essentiality of the four conserved cysteine residues has also been demonstrated in promoting disulfide bond formation in vivo and a mixed disulfide between a cysteine of DsbA of E. coli, and one of the cysteines (Cys(57)) of the VKOR homologue has been identified to be a likely intermediate in the disulfide bond-forming pathway. These studies may inform future resolution of issues surrounding the functioning of human VKOR.

FIG. 1.

Alkaline phosphatase fusions to Mtb VKOR. (A) Positions of the alkaline phosphatase fusions with respect to the topology suggested by homology to S. sp. VKOR. (B) Alkaline phosphatase activities of the fusions measured in single copy in FA113. The activities were normalized to fusion 82, which was given a value of 100. (C) Western blot of MtbVKOR-alkaline phosphatase fusion proteins expressed from plasmids in FA113.

FIG. 2.

Essentiality of the cysteine residues for MtbVKOR function. Cysteine-to-alanine substitutions of each of the cysteines, as well as double mutants of the pairs, were constructed. Motility is dependent on disulfide-bond formation, and an E. coli ΔdsbB strain complemented with wild-type MtbVKOR is motile (top of plate). However, none of the cysteine mutants confers motility to the strain, confirming that they are essential for disulfide-bond formation via MtbVKOR.

FIG. 3.

Growth defect of the cysteine mutants of VKOR in an M. smegmatis vkor deletion strain on minimal medium (7H10). The growth defect is complemented only by expression of wild-type VKOR or His-tagged VKOR. All of the cysteine mutants of VKOR failed to grow on minimal media (7H10).

FIG. 4.

A DsbA-MtbVKOR complex depends on cysteine 57 of MtbVKOR. Overexposure of Western blots of the various MtbVKOR cysteine mutants revealed a complex between DsbA and MtbVKOR. This complex disappears when MtbVKOR is missing cysteine 57 (lanes 3 and 5) (see anti-DsbA blot, because MtbVKOR has a dimeric form that runs at the same position as the DsbA-MtbVKOR complex). Essentially all higher-MW bands are mixed disulfide complexes, as indicated by their disappearance after treatment of extracts with β-mercaptoethanol (β-ME).

FIG. 5.

A comparison of proposed electron transport by MtbVKOR and E. coli DsbB. In both enzymes, the amino-terminal cysteine in one of the loop pairs interacts with DsbA, and all four cysteines are essential for disulfide bond–forming activity.