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Role of dimerization in the catalytic properties of the Escherichia coli disulfide isomerase DsbC.

ABSTRACT: The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two alpha-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly(3)-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.

SUBMITTER: Arredondo SA

PROVIDER: S-EPMC2781991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

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Role of dimerization in the catalytic properties of the Escherichia coli disulfide isomerase DsbC.

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