Three-dimensional motions of GroEL during substrate protein recognition

GroEL is a bacterial chaperonin responsible for the assisted folding of non-native and misfolded polypeptides into biologically active proteins. The adaptive nature of the recognition mechanism of chaperonins toward client polypeptides inherently lends itself to structural heterogeneity, which hampers detailed analyses of intermolecular recognition and binding. In this report, we used single-particle cryo-EM and multiple rounds of focused mask three-dimensional classification to reveal a landscape of distinct snapshots of endogenous GroEL complexed with an unfolded substrate, the water-soluble domain of human UDP glucuronosyltransferase 1A (UGT1A), at 2.7–3.5 Å resolution. We demonstrate that UGT1A occupies the GroEL ring asymmetrically, engaging with 2–3 contiguous subunits and that a subunit bound to UGT1A exhibits a wider range of conformational dynamics, consistent with AlphaFold models. These data reveal molecular motions during initial substrate capture at near-atomic detail.