High-resolution structure determination of substrate-bound and unbound GroEL chaperonin by single-particle cryo-EM.

概要

Bacterial chaperonin GroEL is an ATP-driven molecular machine that captures and assists in folding unfolded or misfolded polypeptides into physiologically active proteins. GroEL has a binary ring decatetramer (14-mer) structure made up of two back-to-back, slightly staggered heptameric rings, each consisting of seven identical protomers (subunits) that are further divided into three key functional domains; apical, intermediate, and equatorial domains. It is generally believed that the GroEL mediated folding reaction cycle proceeds as follows. First, substrate polypeptide is captured by the substrate-binding helices located in the apical domains lining an open ring cavity. Next, ATP binds to the equatorial domain, inducing conformational change that allows recruitment of the co-chaperonin GroES, resulting in the formation of the GroEL/ES complex. The substrate is encapsulated in the enclosed ring where the folding reaction begins, which continues until ATP hydrolysis triggers the release of GroES and native substrate.
　Many studies have investigated structures of GroEL-substrate complexes to gain insights into the precise mechanism of how this molecular machine work. Due to the structural and compositional heterogeneity of the sample, however, it has been difficult to obtain atomic or near-atomic resolution structures of the complex required to facilitate mechanistic understanding of the reaction. Although recent advances in single-particle cryo-electron microscopy (cryo-EM) have ushered in a revolution in structural biology, and this technology can potentially examine compositionally or structurally heterogenous samples, the overall symmetric shape of the GroEL ring with subtle local variations hampered its detailed structural analyses.
　This study presents a high-resolution reconstruction of an endogenous GroEL in complex with a unique substrate UGT1A spontaneously formed in E. coli by single-particle cryo-EM. Using tools and methodologies specifically designed for analyzing particles with local conformational variations, this study demonstrates a landscape of distinct structural snapshots of GroEL heptameric ring with differential substrate occupation states and subunit flexibility at unprecedented resolutions.
　First, all the GroEL-UGTIA complex particles were used to derive all-in, “consensus” reconstruction map at a resolution of 2.8A. Next, successive rounds of tailored mask classification was performed on the consensus map, resulting in three new GroEL-UGTIA complex structures： two minority GroEL-UGTIA complexes where UGT1A was bound to only one ring, reported at 3.26 A & 3.5 A resolutions, and one dominant complex with both rings bound by UGT1A, reported at 2.7 A. From these reconstructions, it was found that UGT1A interacted with 2-3 neighboring subunits at a time and located asymmetrically in the heptameric ring.
　Continued rounds of masked classification over a substrate-bound protomer and an unbound protomer resulted in a series of discrete conformations that constitute a trajectory between the two states, involving a large apical domain elevation movement. Additionally, this elevation coincided with changes in the ATP binding pocket. Flexible docking simulation of ATP to this altered conformation suggests that it has higher nucleotide affinity, pointing toward a putative mechanism by which substrate and ATP binding to GroEL are kinetically coupled. Most importantly, the 2.7 A map visualized clear density for a part of bound UGT1A, offering an unprecedented view of unfolded substrate binding to the apical domain of GroEL near the helix-I, which had been predicted as the substrate binding site. Taken together, these data provide a novel vantage point from which a vast chaperonin-assisted protein folding landscape can be observed.