Naturally Occurring Osmolytes Modulate the Nano-Mechanical Properties of Polycystic Kidney Disease (PKD) Domains

Abstract

Polycystin-1 is a large transmembrane protein, which, when mutated, cause autosomal dominant polycystic kidney disease, one of the most common life-threatening genetic diseases which is a leading cause of kidney failure. PC1 is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. On the other hand, it is known that the native conformation of proteins can be stabilized by protecting osmolytes which are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here we used single-molecule force spectroscopy to directly study the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and TMAO can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated Polycystin-2 channel or other intracellular events mediated by PC1. This work was funded by NIH grant R01DK073394 and the PKD Foundation (grant 116a2r).