Title: Pathogenic Mutations alter the Mechanical Stability of Polycystin-1 Immunoglobulin PKD Domains
Abstract: Mutations in polycystin-1 can cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), which is a leading cause of renal failure. The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary cilia, neighboring cells, and extracellular matrix. polycystin-1 is a large membrane protein that has a long N-terminal extracellular region (about 3000 aa) with a multimodular structure including sixteen Ig-like PKD domains, which are targeted by many pathogenic missense mutations. The mechanical properties of the wild-type PKD domains are known; however, nothing is known about the effects of pathogenic mutations on the biophysical properties of PKD domains. Here we investigated the mechanical properties of several pathogenic missense mutants on the first Ig-like domain (PKD-d1) by using a combination of protein engineering and single-molecule atomic force spectroscopy. We found that missense mutations alter the mechanical unfolding and refolding pathways of PKD-d1 resulting in distinct mechanical phenotypes. Using a simple 2-state energy diagram we find that point mutations can affect the free energy of mechanical unfolding/refolding and the position of the transition state. Depending on the pulling speed we find that a mutation can increase or decrease the unfolding forces. This indicates that the pathogenic mutants can affect the PKD domain's normal response to external mechanical forces and may help us understand the molecular mechanisms underlying ADPKD. This work is funded by NIH (grant R01DK073394) and by the Polycystic Kidney Foundation (grant 116a2r).