Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

Abstract

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.membrane protein assembly | YidC | VKORc1 | anticoagulants

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

“…These strains were used in order to detect weak inhibitors that are not specific to each strain, that is for Pa DsbB1 inhibitors, a strain expressing low levels of Mt VKOR (DHB7657 strain) while for Mt VKOR inhibitors, a strain expressing low levels of Ec DsbB (DHB7935 strain). Given the importance of the human VKOR homolog in the activation of blood clotting factors, we also screened hits with an additional E. coli mutant strain expressing a rat ( Rattus novergicus ) VKOR homolog ( Rn VKOR, FSH250 strain) which conferred upon the strain the ability to oxidize Ec DsbA (Hatahet et al, ). This was done in order to discard compounds that may have an unwanted anticoagulant side effect.…”

“…The conditions used for CL382 strain in X‐Gal plates required the addition of 25 μM of IPTG. FSH250 strain was used with 300 μM IPTG and 60 μg mL –1 of X‐Gal instead of the 120 μg mL –1 used for all the other strains (Hatahet et al, ). P. aeruginosa clean deletion mutants were constructed using overlap extension PCR and homologous recombination as described before (Horton, ).…”

“…In fact, the interactions between the redox‐active cysteines of DsbA and VKOR proceed by the same steps seen between DsbA and DsbB (Wang et al, ). Furthermore, VKORs from other bacteria and one eukaryote can complement an E. coli with a dsbB deletion (Dutton et al, ; Hatahet et al, ).…”

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

“…Additionally, reconstitution of recombinant hVKOR activity from insoluble fractions was strictly dependent on the nature of the membrane composition [80]. Attempts to restore disulfide bond formation to a ΔdsbB strain of E. coli by expressing either the rat or human VKOR have so far been unsuccessful at least in part due to the lack of stable expression [81]. However, selection for a rat VKOR functional in substituting for DsbB in this system yielded a collection of mutants in the rat vkor gene that encoded amino acid changes in the protein.…”

“…The charge distribution of amino acids is known to play an important role in establishing the proper topology of membrane proteins [82,83]. Further, even higher levels of protein are detected when the mutant proteins are expressed in E. coli strains carrying mutations that alter the YidC insertase, a protein necessary for membrane localization of some proteins or mutations eliminating the cytoplasmic protease HslV [81]. Mutant strains harboring both yidC and hslV mutations showed significantly more VKOR activity and protein levels.…”