Pathways of allosteric regulation in Hsp70 chaperones.

Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s.

Functional analysis of Hsp70 inhibitors.

The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES), which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8) is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD) in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD). Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.

Mechanics of Hsp70 chaperones enables differential interaction with client proteins.

Hsp70 chaperones interact with a wide spectrum of substrates ranging from unfolded to natively folded and aggregated proteins. Structural evidence suggests that bound substrates are entirely enclosed in a ß-sheet cavity covered by a helical lid, which requires structural rearrangements including lid opening to allow substrate access. We analyzed the mechanics of the lid movement of bacterial DnaK by disulfide fixation of lid elements to the ß-sheet and by electron paramagnetic resonance spectroscopy using spin labels in the lid and ß-sheet. Our results indicate that the lid-forming helix B adopts at least three conformational states and, notably, does not close over bound proteins, implying that DnaK does not only bind to extended peptide stretches of protein substrates but can also accommodate regions with substantial tertiary structure. This flexible binding mechanism provides a basis for the broad spectrum of substrate conformers of Hsp70s.

Asna1/TRC40-mediated membrane insertion of tail-anchored proteins.

Tail-anchored (TA) proteins insert post-translationally into the membrane of the endoplasmic reticulum (ER) and span the membrane by their C-terminal transmembrane domain. We have reconstituted membrane insertion of TA proteins from recombinant Asna1/TA protein complexes and ER-derived membranes. Our data show that Asna1 can mediate membrane insertion of RAMP4 and Sec61beta without the participation of other cytosolic proteins by a mechanism that depends on the presence of ATP or ADP and a protease-sensitive receptor in the ER membrane. By contrast, membrane insertion of cytochrome b5 can proceed independently of Asna1 and nucleotides.