Disrupted TRiC allostery and conformational equilibrium by LCA-linked mutations: structural and functional insights

Abstract

The eukaryotic chaperonin TRiC/CCT is essential for proteostatis, yet the molecular basis of its subunit-specific pathologies remains poorly understood. Here, we elucidate the molecular mechanism of Leber Congenital Amaurosis (LCA), a severe hereditary retinal dystrophy arised from mutations in the CCT2 subunit of TRiC. By integrating cryo-electron microscopy, biochemistry, and proteomics, we demonstrate that LCA-associated mutations (T400P and R516H in CCT2) disrupt TRiCs critical intra-molecular and intra-ring allosteric network and impair its functional cycle, drastically reducing the population of the folding-active closed state. Unexpectedly, we captured a fully folded endogenous -tubulin within the mutant TRiC chamber, revealing a unique CCT8 C-terminal tail involved folding pathway, distinct from {beta}-tubulin. Furthermore, cellular proteomics revealed that TRiC dysfunction causes a specific downregulation of essential SLC membrane transporters. We propose that the loss of these transporters is likely catastrophic for metabolically demanding tissues like the retina and developing embryo. Our work provides a direct mechanistic link between TRiC structural defects and LCA pathology, offering a new framework for understanding etiology of chaperonopathies.