Structural and Functional Analysis of RPE65 Folding by the Cytosolic Chaperonin CCT

Abstract

A key step in the classical visual cycle is the trans‐to‐cis isomerization of retinoid intermediates by RPE65. Mutations in RPE65 disrupt this process and result in retinal dystrophies such as Leber congenital amaurosis (LCA). Many of these are single‐point missense mutations that occur far from the active site and appear to act by destabilizing the protein structure. Little is known, however, about how RPE65 is folded or how folding is disrupted by mutations. We have evidence that RPE65 folding is assisted by the cytosolic chaperonin CCT. CCT is a large double‐ring complex that is estimated to fold approximately 10% of the proteome and specializes in proteins with complex β‐propeller folds like RPE65. CCT interacts with both WT RPE65 and several LCA‐associated mutants (R91W, L408P, Y368H, R515W). Notably, the interactions between CCT and the mutants are approximately twice as strong as the interaction with the WT despite drastically reduced expression levels, consistent with their decreased stability. In addition, radiolabel pulse‐chase analysis shows that nascent RPE65 associates with CCT and dissociates over time, as expected of a substrate. Taken together, these results suggested that RPE65 is a true substrate of CCT and prompted us to seek a high‐resolution cryo‐EM structure of the complex in order to better understand how CCT folds RPE65. We isolated the complex using a tandem‐affinity approach and have obtained a preliminary 4.2 Å structure which shows the two rings of CCT and a mass attributable to RPE65 sitting between them. In contrast, our previously solved structure of Gβ1 bound to CCT showed Gβ1 interacting the with the apical domains of CCT at the top of the folding cavity. Further improvement of the RPE65‐CCT structure will shed light on mechanistic differences between RPE65 and Gβ1 folding and will provide a basis for understanding how CCT handles the folding defects caused by RPE65 mutations.