C&SP20: Structural plasticity of chaperonins determined by cryo-electron microscopy: Modeling the machinery of GroEL and TRiC

A. Collaborating Investigators: Wah Chiu,¹ and Ivet Bahar

B. Institutions: ¹Baylor College of Medicine, ²University of Pittsburgh

C. Funding Status of Project: P01NS092525 (Chiu, Co-PI) 4/1/2016-3/31/2021; 5P41-GM103832-31 (Chiu) 12/1/96-12/31/19

D. Biomedical Research Problem

Chaperonins are essential mediators of many functions in the cell, including protein folding. These are oligomeric machines, of ~ 1 megadalton, with two back-to-back rings enclosing a central cavity that accommodates polypeptide substrates. The Chiu lab has solved near-atomic resolution cryoEM structures of GroEL from bacteria,³⁷ Mm-Cpn from archaea,³⁸ and TRiC from eukaryotes.³⁹ In addition, they have studied them in different nucleotide-binding states and with a variety of substrates.^40-44 These studies show that chaperonins assume various conformations under different conditions, i.e. structural plasticity is an inherent property of chaperonins.

Recent advances in direct electron detectors used in cryoEM^45,46 have made cryoEM a feasible imaging modality to record images of molecular machines with high quantum detection efficiency and high information content. Fig VIII.3 panel a is a 3.7 Å cryoEM map of GroEL using 40,000 particle images showing unambiguous resolution of backbone and side-chain densities (panel b). The time from data collection to a complete model was less than 3 weeks (Roh and Chiu, unpublished). Using a more advanced image processing method known as focused 3D classification,⁴⁷ we re-analyzed each of the 14 protein subunits in each of the GroEL particle images. We were able to retrieve three types of conformations (Figure VIII.3 c), and determined that they occur randomly in each GroEL machine in variable proportions. Each of them has a characteristic apical domain structure, which coincide with different subunit structures in the PDB (chains B, J and I of PDB ID 1XCK). These findings demonstrate the power of cryoEM to reveal variable conformations of protein components in one single biological machine at a time at near-atomic resolution. A structure-based computational analysis of the dynamics of GroEL as well as TRiC can help further our understanding of the structural mechanisms underlying their function and provide insights into the role of structural plasticity in enabling their allosteric machinery.

E. Methods and Procedures. Our ongoing activity is to determine GroEL cryoEM structure with GroES and carboxylase substrate. In our newly funded program project, we will also pursue the cryoEM characterization of TRiC complexed with mutant huntingtin exon extracted from Huntington's disease (HD) cell model and mouse neuron model. These experiments will provide a mechanistic understanding of how TRiC prevents the progression of aggregation in polyQ,⁴⁴ and mutant huntingtin exon⁴⁸ as part of our effort to develop a TRiC-like reagent for HD therapeutics. The Bahar lab has experience on the machinery and conformational variability of GroEL-GroES,^49-53 which will be further analyzed in the light of the new data generated by the Chiu lab, and with the help of novel extensions of elastic network models (ENMs) developed in TR&D1 subaim 1.1. The computationally predicted dynamics will be compared to the structural variability observed in cryoEM, and the interactions that modulate GroEL allosteric machinery will be identified. Similar methods will be used for TRiC, toward elucidating its spectrum of motions and how its dynamics relates to its chaperoning activities. Target sites whose perturbation may alter/control TRiC machinery will be identified using ProDy tools,⁵⁴ toward assisting in the design of new therapeutic strategies against HD. We anticipate a productive interaction between this project and C&SP16 on the identification of neuroprotectives for HD with Dr. Friedlander (UPMC), an expert in the etiology of HD as well as discovery of neuroprotectives against HD.^55-60