GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome.

The bacterial chaperonin GroEL/ES promotes protein folding post-translation by transiently encapsulating client proteins within a central chamber. GroEL also binds translating ribosomes in vivo, implying an additional role in cotranslational folding. However, how GroEL/ES recognises and modulates ribosome-tethered nascent proteins is unclear. Here, we used biochemical reconstitution, structural proteomics and electron microscopy to study the mechanism by which GroEL/ES engages nascent polypeptides. We show that GroEL binds nascent chains on the inside of its cavity via the apical domains and disordered C-terminal tails, resulting in local structural destabilization of the client. Ribosome-tethered nascent domains are partially encapsulated upon GroES binding to GroEL, and recover their original conformation in the chaperonin cavity. Reconstitution of chaperone competition at the ribosome shows that both Trigger factor and GroEL can be accommodated on long nascent chains, but GroEL and DnaK are mutually antagonistic. Our findings extend the role of GroEL/ES in de novo protein folding, and reveal a plasticity of the chaperonin mechanism that allows cotranslational client encapsulation.