Molecular chaperones are a diverse group of proteins vital to defending cells from the dangers of protein misfolding and aggregation, phenomena implicated in a host of debilitating human conditions. Chaperones perform this protective function by facilitating proper protein folding and assembly, refolding misfolded proteins, preventing and even reversing aggregation, and delivering irreparably damaged proteins for disposal. It is therefore not surprising that genetic mutations in chaperone proteins are implicated in multiple human disorders, including neurodegeneration, myopathies, metabolic disorders, and even cancer. Despite their central role, the large size and intrinsic dynamics of many chaperones have long obscured our understanding of how such mutations affect chaperones’ structure and functions, leading to disease.
Here, we demonstrate how advanced NMR combined with biophysical biochemical assays can unveil the mechanism of chaperone function and decipher the core of their malfunction in disease. We focus on DNAJC12, a ~700 kDa JDP chaperone whose mutations were recently identified in patients with hyperphenylalaninemia (HPA), the most common inherited metabolic disorder. Using NMR, we define the structural features of DNAJC12 and show how it stabilizes its client phenylalanine hydroxylase (PAH) to prevent misfolding. Our data reveal how pathogenic DNAJC12 mutations compromise this chaperone–client cycle, providing a mechanistic link between protein misfolding and the development of HPA. As a second example, we illustrate how disease mutations in the chaperone DNAJB6 reshape its structural ensemble, leading to unregulated recruitment and hyperactivation of Hsp70. This aberrant engagement depletes Hsp70 availability in cells and underlies the molecular basis of Limb Girdle Muscular Dystrophy.