Abstract
Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.
Highlights
Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two wellstructured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker
Extensive work using the Escherichia coli Hsp70 DnaK as a model has shown that when Hsp70 chaperones are bound to ADP, their N-terminal nucleotide-binding domain (NBD) is disengaged (“undocked”) from the C-terminal substrate-binding domain (SBD), the helical lid of the SBD is closed over the substratebinding cleft, and the substrate on/off binding rates to the SBD are slow with high binding affinity for substrates [1,2,3]
Based on inspection of prokaryotic Hsp70 sequences and domain X-ray crystal structures, we defined the DnaK linker region as 384GDVKDVLLLDVT395 because Gly384 terminates a highly conserved ␣-helix in the NBD (PDB code 1DKG) [16], and the residue following Thr395 (Pro396) is the first residue belonging to secondary structure associated with the SBD (PDB code 1DKX) [17]
Summary
Hsp70, 70-kDa heat shock protein; NBD, nucleotide-binding domain; SBD, substrate-binding domain; MD, molecular dynamics; MSA, multiple-sequence alignment; DS, dominant state; PDB, Protein Data Bank. They report the residues that undergo the largest, induced structural perturbations and infer that these are probably critical to allostery Among these were residues Lys387– Val389, Leu391, and Asp393 in the linker, consistent with the present work. The picture that emerges from this analysis presents the interdomain linker as a dynamic switch that performs a key role in interdomain allostery through alternative interactions with either the NBD or the SBD and restricted interdomain relative orientations. Our observations offer an explanation for several evolutionarily conserved features of the interdomain linker
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