RESUMO
We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.
Assuntos
Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inibidores , Chaperonina 10/química , Escherichia coli/efeitos dos fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inibidores , Chaperonina 60/química , Benzoxazóis/química , Benzoxazóis/farmacologia , Benzoxazóis/síntese química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/químicaRESUMO
Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.
Assuntos
Trifosfato de Adenosina , Chaperonina 10 , Microscopia Crioeletrônica , Modelos Moleculares , Ligação Proteica , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Chaperonina 10/metabolismo , Chaperonina 10/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Adenilil Imidodifosfato/metabolismo , Adenilil Imidodifosfato/química , Conformação Proteica , Hydrogenophilaceae/metabolismo , Hydrogenophilaceae/química , Subunidades Proteicas/metabolismo , Subunidades Proteicas/químicaRESUMO
In the densely populated intracellular milieu, polypeptides are at constant risk of nonspecific interactions and aggregation, posing a threat to essential cellular functions. Cells rely on a network of protein folding factors to deal with this challenge. The Hsp60 family of molecular chaperones, which depend on ATP for function, stands out in the proteostasis network by a characteristic structure comprising two multimeric rings arranged back to back. This review provides an updated overview of GroEL, the bacterial Hsp60, and its GroES (Hsp10) cofactor. Specifically, we highlight recent breakthroughs in understanding the intricate folding mechanisms of the GroEL-GroES nanomachine and explore the newly discovered interaction between GroEL and the chaperedoxin CnoX. Despite considerable research on the GroEL-GroES system, numerous questions remain to be explored.