Abstract
Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.
Highlights
The 70-kDa heat shock proteins (Hsp70s) are essential members of the cellular chaperone machinery, assisting protein folding, disaggregation and trafficking, and their malfunction has been associated with a wide variety of diseases, including cancer, neuro-degeneration, allograft rejection and infection [1]
At a protein concentration of 220 μM, 35% of full-length Hsp70 (FL-Hsp70) exists as oligomeric species, this percentage drops to ca. 10% at 70 μM protein (Figure 1A)
With the aim of shedding light on the details of the mechanism of Hsp70 oligomerization, we generated a series of differently truncated variants of human Hsp70, carefully designed to preserve the native structural topology of the full-length protein while displaying different oligomerization propensities
Summary
The 70-kDa heat shock proteins (Hsp70s) are essential members of the cellular chaperone machinery, assisting protein folding, disaggregation and trafficking, and their malfunction has been associated with a wide variety of diseases, including cancer, neuro-degeneration, allograft rejection and infection [1]. Hsp70s are ubiquitously expressed in organisms ranging from Archaea to Homo sapiens and they are among the most conserved proteins in evolution [2]. This characteristic has allowed conclusions from the studies of different Hsp homologues to be extrapolated to the entire Hsp family. Hsp proteins consist of two structurally independent domains: a conserved nucleotide-binding domain (NBD) with ATPase activity, which in H. sapiens Hsp corresponds to residues 1-383, and a more variable C-terminal substratebinding domain (SBD), corresponding to residues 397-641. Recent evidence suggests that this linker might play an active role in the allosteric communication between domains and in the recruitment of the co-chaperone Hsp40 [4,5,6,7]
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