Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays

Abstract

Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound.Biological significance Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics. (C) 2014 Elsevier B.V. All rights reserved.