Abstract
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.
Highlights
Central to the protein folding activity of Hsp[70] 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)
Asp[481], which is located in loop L6,7 in the SBD, has not been investigated so far, it is in a central position within this network
(2) We reveal the existence of two structurally and functionally distinct signal transmission pathways from NBD to SBD and from SBD to NBD. (3) We show that ATP-induced substrate release is more important for the DnaK chaperone activity in vivo than the stimulation of the ATPase activity by substrates
Summary
Central to the protein folding activity of Hsp[70] 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) We dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp[70] DnaK blocked at distinct steps of allosteric communication. Binding of a protein substrate to the SBD and a J-domain protein to the NBD synergistically stimulate the ATP hydrolysis rate by 41,000-fold[23,24,25,26,27] Aspects of this allosteric control mechanism have been worked out through the effort of many laboratories We further show that two pathways of the allosteric mechanism can be distinguished, which allowed us to experimentally reveal their importance for chaperone activity in vitro and in vivo
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