Assembly and biochemical properties of a human chaperone/co-chaperone protein complex

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.