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Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

ABSTRACT: Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroELSR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroELSR-GroES interaction in a controlled manner, we used groES 7, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES7. We generated GroES7 variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroELSR. GroES7 variants with two mutated modules participated in GroELSR-mediated protein folding in vitro. GroES7 variants with two or three mutated modules collaborated with GroELSR to perform chaperone function in vivo: three GroES7 variants functioned with GroELSR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.

SUBMITTER: Illingworth M

PROVIDER: S-EPMC5575113 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

Illingworth Melissa M Ellis Holly H Chen Lingling L

Scientific reports 20170829 1

Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroEL<sup>SR</sup> forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroEL<sup ...[more]

PMID: 28852160

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Project description:As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.

Dataset Information

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

Publications

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

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