The tale of a double-donut and a redox plugin

Details

Supervisors

Collet, Jean-François

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Hsp60 chaperonins are ATP-dependent molecular machines that assist the folding of proteins. Those chaperonins are ubiquitous and are found in all three domains of life. The Hsp60 chaperonin present in Escherichia coli is GroEL; GroEL has been extensively studied since the 1960’s. GroEL functions in cooperation with its co-chaperone GroES; both proteins are essential. Much of the current knowledge on chaperonin-assisted protein folding comes from the work carried out using the GroEL-ES machinery. Yet, several questions regarding how GroEL assist protein folding remain unanswered, such as how GroEL refolds its substrates, or what are the molecular features that make a polypeptide dependent on GroEL for folding. It is also unknown how GroEL deals with endangered substrates. Indeed, unfolded polypeptides can be oxidatively damaged before they reach GroEL, in particular polypeptides that carry sensitive-cysteines residues, which raises the question of how GroEL can assist their folding. CnoX, the first member of the chaperedoxin family, is a protein that combines a redox-protective and a chaperone function. CnoX was recently identified as the first chaperone able to transfer its substrates to GroEL. In addition, unpublished data from my host lab identified CnoX as a protein forming a stable complex with GroEL. This discovery challenges the accepted view that GroEL-ES works in isolation and supports a model in which CnoX functions as a redox-quality control plugin for GroEL. The main purpose of my master thesis was the validation of the CryoEM structure of the GroEL-CnoX complex recently obtained by my host lab. To this end, I used biomolecular and biochemical techniques such as site-directed mutagenesis, co-immunoprecipitation, gel filtration chromatography and western blotting. I engineered several variants of GroEL with mutations in the binding interface with CnoX and tested their ability to interact with CnoX. This method allowed us to identify two GroEL variants with decreased affinity for CnoX. These variants were analyzed to determine whether they maintain their structural integrity and functionality. Altogether, these experiments allowed us to confirm the zone of interaction between GroEL and CnoX as observed in the CryoEM structure.