Structural interplay of the redox co-chaperone CnoX to GroEL/ES chaperonin.

Protein folding by the bacterial chaperonin GroEL/ES relies on ATP-driven conformational cycles that promote substrate encapsulation and folding. Under oxidative stress, the redox-active co-chaperone CnoX protects oxidized proteins and associates with GroEL, yet the structural basis of its interaction with the GroEL/ES remains incompletely understood. Using single-particle cryo-electron microscopy, we resolved four distinct nucleotide-bound conformational states of CnoX-associated GroEL/ES complexes. CnoX remains tethered to GroEL through its C-terminal TPR domain despite substantial rearrangements of the GroEL apical domains. We further captured a GroEL/ES-CnoX ternary assembly in which CnoX and GroES simultaneously occupy the same GroEL ring, demonstrating that their binding sites are structurally distinct and non-overlapping. Comparison of two GroES-bound states reveals how apical-domain compaction occludes the CnoX-binding surface and coincides with loss of CnoX from the cis-ring. Together, these structures define how CnoX is accommodated and excluded across distinct GroEL/ES conformations and provide a structural framework for understanding the interplay between redox co-chaperones and chaperonin assemblies.