Crystal Structure of the C-terminal 10-kDa Subdomain of Hsc70

Abstract

The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions. They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain. The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain. Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 Å. Its helical region adopted a helix-loop-helix fold. This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70. Moreover, in the crystalline state, the 10-kDa subdomain formed dimers. The results of gel filtration chromatography further supported the view that this subdomain was selfassociated. Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers. These findings suggest that the ␣-helical region of the 10-kDa subdomain dictates the chaperone self-association.

A Disulfide Bridge Mediated by Cysteine 574 Is Formed in the Dimer of the 70-kDa Heat Shock Protein

“…However, there was an apparent discrepancy as to the role of sub-domains, whether the client-binding sub-domain or the lid sub-domain is critical for this role. The 10-kDa C-terminal lid sub-domain (amino acids 542-646) of rat Hsc70, which forms an elongated bundle-like structure, dictates the self-association (22). In contrast, Fouchaq et al (20) reported that the 17-kDa peptide-binding sub-domain (amino acids 385-540 of Hsc70) was involved in the oligomerization process.…”

“…According to the structure proposed by Chou et al (22), the lid sub-domain of one molecule associates with that of another molecule in an anti-parallel manner, in which 2 cysteines are separated from each other, and therefore, do not seem to form a disulfide bridge. Thus, we insist that the dimeric structure proposed by Chou et al (22) should be carefully re-investigated.…”

“…Chou et al (22) reported the crystal structure of the C-terminal 10-kDa lid sub-domain of rat Hsc70. The lid sub-domain formed a package of 48 molecules in a unit cell.…”

“…In contrast, Chou et al (22) proposed that the 10-kDa lid sub-domain of Hsc70 is responsible for the dimerization and oligomerization. Therefore, it remains unknown as to whether the client-binding sub-domain or the lid sub-domain is critical for the dimerization.…”

Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B′, in differentiated human neuronal cells

“…Structural studies to pinpoint the region involved in selfassociation have focused on Hsc70 (Angelidis et al 1999;Fouchaq et al 1999;Chou et al 2003;Nemoto et al 2006;Amor-Mahjoub et al 2007). The regions essential for selfassociation of Hsc70 were found to be the conserved hydrophobic residues within the interdomain linker region and the helices at the end of the C terminus (AmorMahjoub et al 2007).…”