Abstract

All cells depend on properly folded proteins for survival and function. Misfolding of proteins results in loss of critical functions and may trigger the misfolding of other nearby proteins leading to toxic aggregation. While many proteins can fold on their own, others with complicated domain structures require assistance from protein folding machines called chaperones. The most complex and highly specialized of all chaperones is the eukaryotic chaperonin complex CCT which is necessary for the folding of a wide variety of essential proteins. These include the cytoskeletal proteins actin and tubulin as well as the Gβ subunit of the G protein heterotrimer. However, CCT activity can also drive diseases such as cancer and viral infections and could represent a high value therapeutic target if the mechanisms by which it folds its different client proteins were better understood. To this end, we identified and characterized an interaction between CCT and the RNA-dependent RNA polymerase of SARS-CoV-2, the virus responsible for the deadly global pandemic that began in 2019. We showed that SARS-CoV-2 replication is impaired by loss of CCT and that the polymerase, designated Nsp12, interacts with CCT upon synthesis and quickly releases - the hallmark pattern of a CCT substrate. Furthermore, we solved a 3.3 Å cryo-EM structure of Nsp12 bound to open CCT showing Nsp12 binding between the two rings of CCT and extending up through of the folding chambers and out of CCT. At 107 kD, Nsp12 is the largest substrate ever visualized inside of CCT and answers a long-standing question in the field of how CCT could accommodate substrates larger than its 70 kD folding chamber. Given that CCT is known to fold proteins with WD40-repeat domains, we also investigated a potential relationship between CCT and RPE65. RPE65, which contains a WD40 domain, is the retinyl ester isomerase that converts all-trans retinyl esters into 11-cis retinol, a key step in the visual cycle, and its mutation is a common cause of hereditary retinal dystrophies. We showed that CCT interacts with RPE65 and that nascent RPE65 binds and releases from CCT albeit with relatively slow kinetics. However, RPE65 is not dependent on CCT for expression or activity. This suggests that the relationship between RPE65 and CCT may represent a novel CCT function distinct from the canonical obligate substrate dynamic.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

https://lib.byu.edu/about/copyright/