This study has revealed new insights into the fundamental mechanism of allostery of a paradigmatic Hsp70, DnaK ( and Figure S6
). We find that the Hsp70 allosteric landscape comprises three distinct protein conformations (): undocked (ADP-bound) and docked (ATP-bound) ‘end-point’ states, and a previously unidentified intermediate, the allosterically active, domain-dissociated linker-bound conformation that is partially populated in the presence of ATP and substrate. Each conformation is characterized by different arrangements of Hsp70 allosteric structural elements (NBD, β-SBD, α-helical lid), while two flexible regions–the interdomain linker and helix B of the helical lid–provide adjustable coupling connections between these units and create tunable interfaces between the structural elements (). This ‘Lego®
’-like architecture creates a set of thermodynamic linkages that provide an explanation for the fundamental mystery of Hsp70 allostery: how events at each of the two domains can influence the other domain.
Allostery in Hsp70s is achieved because binding of nucleotide and substrate ligands are thermodynamically linked so as to control the conformations of individual domains. In order for this allosteric machine to function, each separate domain must possess the capacity to sample (at least) two distinct conformations (): The result of linkages between domains is a novel state endowed with the properties required for active allostery (Csermely et al., 2010
; del Sol et al., 2009
; Smock and Gierasch, 2009
): the ability to ‘breathe’ and sample multiple local conformations, including one with a catalytically active array of nucleotide ligands, and one with an un-lidded, disturbed substrate-binding site, which should have fast and reversible substrate binding/release. In the allosteric cycle of an Hsp70 depicted in , this state corresponds to the obligatory intermediate between the two end-point ADP- and ATP-bound states. For the isolated NBD, the binding of ATP perturbs the intradomain conformation so as to favor linker-binding and high ATPase activity (Bhattacharya et al., 2009
; Revington et al., 2004
; Zhuravleva and Gierasch, 2011
). In full-length DnaK this ATP-induced linker binding transmits a signal to the SBD via stabilizing interactions between the NBD and SBD. Note that only minor changes in reorientations of NBD subdomains drastically affect ATPase activity of the protein (Zhuravleva and Gierasch, 2011
), which explains how interactions between the linker and the NBD (in the allosterically active, domain-undocked conformation) and between the NBD and the SBD (in the docked conformation) significantly affect ATPase activity. Binding of substrate is coupled to these NBD conformational changes because of its direct stabilizing effect on the β-SBD-α-helical lid interface, and indirect destabilizing effect on the NBD-β-SBD interface. For the SBD, only one of its conformations has been described at atomic resolution. However, our results clearly demonstrate that domain docking stabilizes a very different un-lidded β-SBD conformation that we know has a markedly reduced capacity to bind substrates.
The delicate balance among conformational states created by thermodynamic coupling of opposing energetic contributions leads to exquisite ‘tunability’ of the Hsp70 system (Figure S7E
). Each ‘end-point’ Hsp70 state is stabilized by only one major intrinsic interaction (): either the β-SBD–α-helical lid interaction in the undocked state (in the presence of ADP and substrate) or the NBD–β-SBD interaction in the docked (ATP-bound) state, while both interactions contribute to the allosterically active (ATP- and substrate-bound) conformation. Consequently, even minor perturbations of these interfaces result in redistributions in the Hsp70 conformational ensemble, and (through resulting ATPase activity and substrate affinity) define kinetics and thermodynamics of the Hsp70 allosteric cycle. Thus, the conformational distribution underlying the Hsp70 allosteric cycle can be readily shifted by either internal (sequence changes) or external factors (binding to co-chaperones, other chaperones, and different substrates).
The energetic tug-of-war in Hsp70s between intradomain interactions and interdomain interfaces provides an explanation for a number of previous observations, including the observation of a marked decrease in substrate affinity upon perturbation of the β-SBD–α-helical lid interaction (Fernandez-Saiz et al., 2006
; Moro et al., 2004
) and the fact that ATPase stimulation is proportional to substrate affinity (Mayer et al., 2000
). Alterations in substrate binding affinity or kinetics clearly alter the allosteric reaction propensities. As a result, the behavior of the Hsp70 can be tuned to individual substrates, depending on their folding and aggregation properties, or on the physiological situation. DnaK is a ‘hub’ among chaperone networks, and forms complexes with at least 700 substrates (Calloni et al., 2012
): Its tunability enables it to perform its allosteric cycle differently, depending on these extrinsic factors. Moreover, binding to a large substrate will significantly destabilize the interaction between the β-SBD and the α-helical lid (Schlecht et al., 2011
), providing yet another way to affect the Hsp70 ensemble and result in substrate-dependent modulation of Hsp70 function.
Co-chaperones serve as extrinsic contributors to the allosteric balancing act in Hsp70s. We speculate that co-chaperone effects on Hsp70s will be clarified in terms of the balance of intra- and interdomain interactions. For example, shifting of the equilibrium between the linker-bound and linker-unbound conformations likely underlies the ability of the DnaJ-class of co-chaperone to enhance Hsp70 ATPase activities (Jiang et al., 2007
). A recently discovered dynamic interface between DnaJ and DnaK (the segment 206-221 of the NBD) (Ahmad et al., 2011
) overlaps with the NBD–SBD interdomain interfaces and provides another means to regulate Hsp70 ATPase activity.
The ‘tunability’ of the Hsp70 system offers an explanation for the striking functional diversity in the Hsp70 family (Kampinga and Craig, 2010
; Sharma and Masison, 2011
). Evolutionary tuning can occur via sequence changes at the key coupling interfaces. As illustrated above (), even single conservative amino acid changes shift the equilibrium among docked, undocked (linker-unbound) and allosterically active (linker-bound) states and thus ‘tune’ conformational distributions to adjust kinetics and thermodynamic of the allosteric cycle to specific substrates, environment and function in different Hsp70 members. It will be of great interest to further explore the impact of sequence variations in these key interfaces among the Hsp70 family.
Taken together, our results provide new insights into the mechanism of Hsp70 allostery that explain many previous experimental observations, elucidate the basis of the striking functional diversity within the Hsp70 family, and reveal ‘tunable’ allosteric segments in Hsp70, which comprise potential binding sites for Hsp70 co-chaperones. The new insights into ‘tunability’ also provide a basis for design of small allosteric modulators of Hsp70 function, which are shown to have the great potential for therapeutic targeting of the Hsp70 system (Chang et al., 2011
; Rousaki et al., 2011
). Our data on Hsp70s also have implications more broadly, as allostery in other systems is likely to exploit analogous ligand-modulated changes in thermodynamic linkages between protein domains and allosteric interfaces. From an evolutionary standpoint, it is clear from the Hsp70 system that linking conformational equilibria within domains via interdomain interfaces is a blueprint to create allosteric signaling in multidomain protein systems. Indeed, recent work from the Ranganathan lab has illustrated successful creation of allosteric signaling by combining otherwise non-allosteric proteins (Halabi et al., 2009
). We believe that the same mechanistic principles harnessed in the two-domain Hsp70s can also be extended as a general allosteric mechanism for another multidomain protein systems and for protein complexes with coupled allosteric functions.