Abstract
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:ES1 complexes has been extensively reported, the substrate interactions of another functional complex, the football-shaped GroEL:ES2, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES2 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:ES2 can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES2 complex as a functional state to assist the substrate folding with visualization.