Cryo-EM structures of GroEL:ES with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin.

The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES complexes with a chemically denatured substrate, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Our structures demonstrate that native-like folded RuBisCO density is captured at the lower part of the GroEL chamber and that GroEL's bulky hydrophobic residues Phe281, Tyr360, and Phe44 contribute to direct contact with RuBisCO density. In addition, our analysis found that GroEL:ES can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES complex as a functional state to assist the substrate folding with visualization.