Assembly and biochemical properties of a human chaperone/co-chaperone protein complex
Creators: name: Vydyanath, Anupama
Publisher: name: University of Westminster
Year: 2010

description: The 70kDa members of the heat shock protein family (eg. Hsp70) function as molecular chaperones by binding to exposed hydrophobic patches on nascent polypeptides forming non-covalent interactions, thereby preventing their aggregation and facilitating their proper folding. The folding reaction comprises of cyclic binding and release of the unfolded substrate powered by ATP hydrolysis. Hsp70 requires the assistance of a co-chaperone, generally provided by the Hsp40 group of proteins, for the cycle of protein folding. Biochemical analyses have mapped the possible sites of interaction between the Hsp70 and Hsp40 proteins and predicted a bipartite mode of interaction between Hsp70 and Hsp40. However, structural investigations into the mechanistic features of the folding cycle have been hampered by the transient nature of interaction. The underlying theme for this work was to therefore structurally understand the assembly of Hsp70 proteins with the Hsp40 co-chaperones as a complex during the Hsp70-assisted folding cycle. This study was carried out using human Hsc70 and HSJ1b as representatives of the Hsp70 and Hsp40 families respectively. The first step to understand this co-operation was to develop a strategy to isolate a complex of Hsc70 and HSJ1b suitable for structural studies. Previous studies have reconstituted the Hsp70/Hsp40 complex in vitro by combining the two proteins in molar ratios in the presence of ATP. In this work a co-expression system was developed and a recombinant form of the human Hsc70/HSJ1b complex was successfully purified using a bacterial expression system. Biochemical characterisation revealed that this chaperone complex can protect ~85% of substrate protein from thermal aggregation. Gel filtration analysis revealed that the complex was composed of a heterogenous mix of ~220 kDa and hetero-oligomeric co-polymer species. Analytical ultracentrifugation confirmed that these hetero-oligomeric co-polymer species were not aggregates, and molecular weight for this species was estimated to be 1.1 MDa. These two species represent potentially two different states of association between Hsc70 and HSJ1b. ATP and heat treatment at 42oC with luciferase were identified as factors which promote the conversion of the oligomeric Hsc70/HSJ1b species to the ~220 kDa Hsc70/HSJ1b species. Domain variants of Hsc70 were then generated and their ability to complex with HSJ1b was investigated. Using these Hsc70 domain variants, the region on Hsc70 paramount for polymerisation was identified. The C-terminal 10 kDa lid region was found to be essential for the chaperone/co-chaperone interaction, since the removal of this zone alters binding, function and conformational properties of the Hsc70 and HSJ1b interaction. X-ray crystallography studies on the full length and the domain complexes were carried out, leading to the structure of the apo form of the nucleotide binding domain of Hsc70. Preliminary electron microscopy (EM) analysis was undertaken of the recombinant Hsc70/HSJ1b complex. The preliminary results from negative staining revealed mostly circular particles and were extremely encouraging. Currently work is being carried out to improve sample homogeneity, which will facilitate further EM studies. Thus the recombinant complex generated in this study is an attractive tool to further our understanding of the functional and structural features of the interactions of Hsp70 with Hsp40.