Structural Transitions in Ancient Chaperonins Illustrate Pathways to Protein Complexity

Chaperonins are essential protein-folding machines that can be categorized into three groups based on their structure and phylogeny: Group I (bacterial GroEL), Group II (archaeal thermosome and eukaryotic CCT), and Group III (bacterial thermosome-like). We used ancestral sequence reconstruction (ASR) and protein resurrection (PR) to infer and experimentally test ancestral chaperonins (ACs) from the last common ancestors of these groups (ACI, ACII, and ACIII). The reconstructions exhibited ATPase activity (except for ACII) and client protein heat protection. Electron microscopy and Cryo-EM showed that ACI formed single 7-mer rings, while ACII formed a mix of single/double 8-mer rings, indicating a gradual evolution from simple single-ring to more complex double-ring structures. We also found a unique cochaperonin-independent closing mechanism in ACII. Together, our results illustrate the early stages of molecular complexity evolution and demonstrate how ancestral proteins can inform models of early cellular life.