Molecular chaperone proteins play a pivotal role in maintaining normal proteostasis within the cell by aiding the correct folding of nascent chains. Understanding how these proteins function, has implications for a milieu of human diseases. Here the structures of Hsc70 from two different species (bovine and porcine), are analysed. These proteins differ by one amino acid in the nucleotide binding domain, M93V (bovine to porcine) and by four residues in the C-terminus, where there is an additional GGXP motif in bovine Hsc70 protein. Small angled X-ray scattering on native Hsc70 found that these proteins varied in their overall size, in both ATP- and ADP-bound conditions. These results indicated that the C-terminus is important for the overall structure and function of Hsc70 protein.
The alternate isoform of Hsc70, Hsc54, has a deletion of residues 464 - 616, resulting in an incomplete SBD, the C-terminus however is still retained. In an effort to glean more information about the C-terminus a recombinant clone of Hsc54 was constructed, along with a bacterial expression cell line that does not contain the Hsc70 prokaryotic homolog protein, DnaK. Recombinant Hsc54 was successfully purified and small angled X-ray scattering studies were undertaken.
These results when amalgamated together identified the C-terminal tail of Hsc70 as an important unfolded protein domain. The small angled X-ray scattering models showed that the GGXP motif influenced the overall compactness of Hsc70 protein. Circular dichroism experiments on chemically synthesised C-terminal tail peptides revealed a temperature-dependent conformational change; a random coil conformation to a poly proline type II helical structure. These results gave rise to a thermal switch hypothesis, whereby upon heat shock the C-terminal domain of Hsc70 transitions from a largely unstructured domain to a loose helical structure, preventing co-chaperones from binding. Leaving the co-chaperones available to bind to the inducible Hsp70 chaperones, which do not contain any GGXP motifs in their C-terminal domain.
