Backbone NMR resonance assignment of Sis1, a type B J-domain protein from Saccharomyces cerevisiae.

Abstract

J-domain proteins (JDPs) are essential cochaperones of heat shock protein 70 (Hsp70), as they bind and deliver misfolded polypeptides while also stimulating ATPase activity, thereby mediating the refolding process and assisting Hsp70 in maintaining cellular proteostasis. Despite their importance, detailed structural information about JDP‒Hsp70 complexes is still being explored due to various technical challenges. One major challenge is the lack of more detailed structural data on full-length JDPs. Class A and B JDPs, the most extensively studied, are typically dimers of 300-400 residue polypeptides with central intrinsically disordered regions. These features complicate structural analysis via NMR and X-ray crystallography techniques. This work presents the ¹H, ¹⁵N, and ¹³C backbone resonance assignments of the full-length (352 residues long) Sis1, a dimeric class B JDP from S. cerevisiae. Our study achieved 70.5% residue assignment distributed across the entire protein, providing probes in all Sis1 domains for the first time. To overcome this challenging task, strategies such as deuteration and 3D BEST-TROSY correlation experiments were used. The methods and results are detailed within the text. We are confident that this achievement will significantly benefit both the structural biology and the proteostasis scientific communities.