Graduate Research Assistant Brigham Young University Provo, Utah, United States
Theresa Smith (Brigham Young University)| Kristen Read (Brigham Young University)| Peter Shen (University of Utah)| Michael Stewart (University of Utah)| Philip Kiser (University of California, Irvine)| Barry Willardson (Brigham Young University)
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 the cytosolic chaperonin CCT plays a role in this process. 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. RPE65 is capable of binding to CCT and nascent overexpressed RPE65 binds and slowly releases from CCT in a radiolabel pulse chase setting. However, in contrast to what has been observed for other canonical CCT substrates, depletion of CCT from cells does not appear to affect either RPE65 expression or its isomerase activity, suggesting that CCT is not required for RPE65 folding. Notably, several LCA-associated RPE65 mutants (R91W, L408P, Y368H, R515W) interact with CCT with approximately twice the affinity of the wild type (WT), despite their drastically reduced expression levels. This indicates that CCT may function to bind and sequester distressed RPE65 molecules. In order to better understand the nature of the interaction between CCT and RPE65, we turned to high-resolution cryo-EM. We isolated the complexes of WT and R91W RPE65 respectively with CCT using a tandem-affinity approach and have investigated their structures by high-resolution cryo-EM. A preliminary 4.2 Å structure of the WT complex shows the two rings of CCT and a mass attributable to RPE65 sitting between them. Once completed, we expect analysis and comparison of these two structures to shed light on why CCT binds RPE65 and how mutations in RPE65 produce the phenotypes associated with LCA.