Insights into the molecular mechanism of allostery in Hsp70s

Abstract

Hsp70s chaperone an amazing number and variety of cellular protein folding processes. Key to their versatility is the recognition of a short degenerate sequence motif, present in practically all polypeptides, and a bidirectional allosteric intramolecular regulation mechanism linking their N-terminal nucleotide binding domain (NBD) and their C-terminal polypeptide substrate binding domain (SBD). Through this interdomain communication ATP binding to the NBD and ATP hydrolysis control the affinity of the SBD for polypeptide substrates and substrate binding to the SBD triggers ATP hydrolysis. Genetic screens for defective variants of Hsp70s and systematic analysis of available structures of the isolated domains revealed some residues involved in allosteric control. Recent elucidation of the crystal structure of the Hsp70 homolog DnaK in the ATP bound open conformation as well as numerous NMR and mutagenesis studies bring us closer to an understanding of the communication between NBD and SBD. In this review we will discuss our current view of the allosteric control mechanism of Hsp70 chaperones.

Keywords: Hsp70 heat-shock proteins; allostery; conformational dynamics; interdomain communication; structure-function relationships.

Figure 1

Conformational cycle of Hsp70s. (A) Structural changes associated with the ATPase cycle of E. coli DnaK. Left, crystal structure of DnaK in the ATP bound open conformation (low-affinity state, PDB ID 4B9Q, Kityk et al., 2012) in cartoon representation with NBD subdomains IA, IB, IIA, IIB in different shades of green, SBDβ in dark red and SBDα in orange. Right, solution structure of DnaK in the ADP bound/nucleotide-free state as derived from residual dipolar coupling NMR experiments and crystal structures of the isolated domains (high-affinity state, PDB IDs 2KHO, Bertelsen et al., 2009) colored as in the ATP bound state and NBDs in identical orientation. (B) Overlay of the crystal structures of DnaK·ATP (PDB ID 4B9Q; gray) and Sse1 (PDB ID 2QXL, Liu and Hendrickson, 2007); NBD, deep teal; SBDβ, cyan; SBDα, blue. (C) Overlay of the crystal structures of DnaK·ATP (PDB ID 4B9Q; gray) and bovine Hsc70(1-554) (PDB ID 1YUW, Jiang et al., 2005) NBD, deep teal; SBDβ, cyan; SBDα, blue.

Figure 2

ATP-induced changes in NBD and SBD and allosteric cycle of Hsp70s. (A) Overlay of the NBD of E. coli DnaK in the ATP bound open conformation (PDB ID 4B9Q, Kityk et al., ; green) and of DnaK in the nucleotide-free/ADP bound state (PDB ID 2KHO, Bertelsen et al., ; blue) in tube representation. Left, standard view; right, only subdomains IA and IB rotated by 120° as indicated. (B) Structure of DnaK in the ATP bound open conformation with residues known to be involved in interdomain communication and found in the NBD-SBD interface in space-filling representation with carbon atoms in yellow, oxygen atoms in red and nitrogen atoms in blue. (C) Overlay of the SBD of DnaK in the ATP bound open conformation; SBDβ, dark red; SBDα, orange and cut for space reasons and the structure of the isolated SBD in complex with a substrate peptide (PDB ID 1DKX, Zhu et al., 1996); SBDβ, cyan; SBDα, dark blue; peptide in light blue and stick representation. Arrows indicate ATP-induced changes visible in this orientation. (D) Overlay of SBDβ as in (A), but rotated by 90° as indicated. Arrows indicate ATP-induced narrowing of the central substrate binding pocket. (E) Overlay of the SBDβ of the two available structures of DnaK in the ATP bound open conformation (PDB IDs 4B9Q, Kityk et al., ; dark red; 4JN4, Qi et al., ; green). Indicated are the substrate enclosing loops L_{1, 2}, L_{3, 4}, and L_{5, 6}. (F) In the ADP state Hsp70s are in equilibrium between the closed conformation with NBD (green) and SBD (dark red) only connected via the conserved interdomain linker (black) and substrate (S) tightly enclosed in the substrate binding pocket and a very transient open conformation with NBD and SBDβ (dark red) docked. Since the open conformation is very transient, substrates only dissociate from this state at low rates. Nucleotide exchange factors (NEFs) catalyze ADP dissociation. Subsequent ATP binding to Hsp70 induces rotation of the NBD lobes toward each other, opening of the lower cleft of the NBD, insertion of the conserved interdomain linker, and docking of SBDβ to the NBD, resulting in opening of the α-helical lid (SBDα, magenta) and release of the substrate with high rates. In the ATP state Hsp70s are also in equilibrium between the open and very transient closed conformation. The outer loops of the SBDβ are highly dynamic. Substrates associate with J-domain proteins (JDP) and bind with high rates to the open conformation of Hsp70. Substrate binding induces closing of the SBDα and dissociation of the SBDβ from the NBD, which allows rotation of the NBD lobes to a position optimal for ATP hydrolysis. Substrates stimulate ATP hydrolysis through a distinct pathway (blue) involving a trigger on the NBD (orange). How JDPs act in synergism with substrates is currently not known. Dashed arrows indicate domain movement/dynamics.