The ATPase domain consists of two large globular subdomains (I and II), each further divided into two small subdomains (A and B; fig. ). The subdomains are separated by a deep cleft at the bottom of which the nucleotide binds in complex with one Mg2+
and two K+
ions contacting all four subdomains (IA, IB, IIA, IIB) [45
]. The X-ray structures of the bovine Hsc70 ATPase domain complexed with several adenosine nucleotides (ADP, AMPPNP, ATP to mutant proteins) revealed that the adenosine nucleotide is positioned in the active site by interactions with two β
- and γ
-phosphate-binding loops and a hydrophobic adenosine binding pocket [45
]. More recent nuclear magnetic resonance (NMR) investigations demonstrate a high flexibility of the ATPase domain with a shearing and tilting motion of the different subdomains towards each other, leading to an opening and closing of the nucleotide binding cleft (fig. ) [46
]. A continuous opening and closing of the nucleotide binding cleft was proposed earlier to depend on the nucleotide present in the nucleotide binding site with the opening frequency being largest in the nucleotide-free state and decreasing in the order nucleotide-free > ADP > ADP+inorganic phosphate > ATP [47
]. These observations explain the different dissociation rates for the different nucleotides and the influence of inorganic phosphate on these dissociation rates (see below).
Genetic and biochemical evidence clearly demonstrates that ATP hydrolysis is essential for the chaperone activity of all Hsp70 proteins tested so far. ATP hydrolysis is the ratelimiting step in the ATPase cycle of most Hsp70 proteins investigated except E. coli
HscA and T. thermophilus
DnaK. The rates of hydrolysis are very low in the ground state, ranging between 3·10−4
, respectively [48
]. The hydrolysis of ATP triggers the closing of the substrate binding cavity and the locking-in of associated substrates. In a thermodynamically coupled process, substrates stimulate the hydrolysis of ATP by typically 2–10-fold. This stimulation by substrates is too low to drive the functional cycle of Hsp70 chaperones. Instead, the activity of co-chaperones of the family of JDPs is required for productive tight coupling of ATP hydrolysis with substrate association [49
]. J-domain co-chaperones are a heterogeneous class of multidomain proteins which share a conserved stretch of approx. 70 residues often located at the N-terminus (fig. ). This so-called J-domain is essential for the interaction of JDPs with their Hsp70 partner proteins. The mechanism of JDPs is best understood for the E. coli
Figure 3 DnaJ and Bag-1 protein families. (a) Domain structure of the 3 DnaJ subfamilies. The different domains are marked in the following way: J, J-domain; G/F, Gly-Phe rich region; Zn, Zn2+ binding domain, C, C-terminal domain of homology. (b) Secondary structure (more ...) E. coli
DnaJ and protein substrates synergistically stimulate the rate of ATP hydrolysis by >1000-fold [49
]. The action of DnaJ requires both binding of protein substrates to the central hydrophobic pocket of DnaK’s substrate binding cavity and structural coupling between DnaK’s ATPase and substrate binding domains, which transmits the substrate binding event to the catalytic center [51
]. Furthermore, DnaJ's coupling activity requires the ability to interact with DnaK through its J-domain [49
] and with substrates through its C-terminal substrate binding domain. DnaJ interacts with substrates in a rapid and transient fashion [54
On the basis of the available data a model for the action of DnaJ has been proposed [49
]. Accordingly, DnaJ starts the functional cycle of the DnaK system by rapid and transient association with the substrate. The substrate can then be transferred onto DnaK in a two-step process involving the transient interaction of the J-domain with DnaK·ATP and the association of the substrate with the open substrate binding cavity of DnaK. Through an interdomain communication, the association of substrates and interaction with the J-domain lowers the activation energy for the hydrolysis of ATP by DnaK.
The synergistic stimulation of ATP hydrolysis by substrates and JDPs has also been observed in other Hsp70 systems, including the action of auxilin and clathrin on Hsc70, of Sec63 and substrates on BiP and of HscB and IscU on HscA [50
]. Thus, the coupling activity of DnaJ proteins appears to be a mechanism that is conserved at least in some Hsp70 chaperones.
The next step in the ATPase cycle, the release of ADP and Pi, allows the subsequent rapid binding of ATP and, consequently, the release of bound substrates and re-establishment of the starting point of the chaperone cycle. Under physiological conditions, i.e. high cytoplasmic ATP concentrations, nucleotide dissociation is rate limiting for substrate release. Nucleotide dissociation is thus a crucial step in the cycle, and it is therefore not surprising that this step is highly regulated and subject to strong evolutionary variation.
The dissociation of bound nucleotides requires an opening of the nucleotide binding cleft. Such movement has been observed for E. coli
DnaK and bovine Hsc70 when their respective nucleotide exchange factors, GrpE and Bag-1, are bound. In the absence of such factors, the equilibrium between the closed and the opened states is probably the determining factor for the intrinsic rates of nucleotide dissociation. For DnaK, the ADP dissociation rates are very low, being 0.004–0.035 s−1
and 0.0004–0.0014 s−1
in the presence and absence of Pi
, respectively [48
]. Although the ADP dissociation rate is 2–60-fold faster than the rate of hydrolysis in the unstimulated cycle, it nevertheless becomes rate limiting when the hydrolysis of ATP is stimulated by DnaJ and substrates. For bovine Hsc70, the dissociation rates for ADP±Pi
are about 20-fold higher than those of DnaK [48
]. Even more dramatic differences exist for HscA, which has dissociation rates for ADP±Pi
that are 700-fold higher than those of DnaK [59
What is the structural basis for these strong kinetic differences between Hsp70 homologs in nucleotide dissociation? Although the modeled ATPase domain structures of DnaK, HscA and Hsc70 are almost completely superimposable, they show subtle differences which allow classification of the entire Hsp70 family into three subfamilies with E. coli
DnaK, E. coli
HscA and human Hsc70 as prototypes [59
]. First, variations exist in an exposed loop in subdomain IIB near the nucleotide binding cleft. DnaK subfamily members share a particularly long loop (A276-R302 in E. coli
DnaK) with subfamily-specific sequence. Members of the Hsc70 subfamily share a loop with subfamily-specific sequence whose tip is 4 residues shorter, while members of the HscA subfamily share a loop that is 10 residues shorter and less conserved in sequence. Second, variations exist in the interface of the nucleotide binding cleft. DnaK proteins contain a hydrophobic patch (L257-V59 of DnaK) at the top of the cleft and two putative salt bridges (E264-R56, upper; E267-K55, lower) that are mainly responsible for the polarity of the interface. In contrast, Hsc70 proteins lack a DnaK-like hydrophobic patch and the upper salt bridge, while HscA proteins lack all three elements.
The loop and the salt bridges constitute a device that allows rapid association of ATP and slow dissociation of ATP and ADP±Pi
. The salt bridges together with the hydrophobic contact probably function in a mouse-trap-like fashion to allow tight closure of the nucleotide binding cleft. The role of the loop is less obvious. Given the considerable size of its exposed part, it has been speculated that it may reach over to subdomain IB of the ATPase domain of DnaK, thereby acting as a latch [59
]. The large variability within the Hsp70 family in nucleotide exchange rates is likely to contribute to the functional diversification of Hsp70 chaperone systems.
The differences between Hsp70 homologs in nucleotide dissociation go along with differences in the regulation of this step. DnaK homologs that have the described trap to prevent nucleotide dissociation require the nucleotide exchange factor, GrpE, for chaperone activities in vivo and in vitro. HscA homologs which lack this device and have high nucleotide dissociation rates neither interact with GrpE nor appear to have their own exchange factor [59
]. Mammalian Hsc70 homologs that have intermediate dissociation rates possess chaperone activity without exchange factor. However, they can interact with a nucleotide exchange factor, Bag-1, perhaps to fulfill special chaperone activities. The two known nucleotide exchange factors for Hsp70 proteins, GrpE and Bag-1, have entirely different structures and mechanisms, although they generate the same conformational open state of their target chaperone [60
]. These proteins may therefore have been generated by convergent functional evolution.
GrpE has a molecular weight of 22 kDa and forms a stable dimer in solution. The X-ray structure of GrpE in complex with the ATPase domain of DnaK was solved to 2.8 Å resolution [60
]. The N-terminus of the truncated GrpE monomer constitutes an α
-helix with the length of 100 Å. The N-terminal helix is connected over a rather unstructured loop to two additional short α
-helices and a small β
-sheet domain composed of six short β
-strands. In addition to the N-terminal helix, the dimer interface is formed by the two short helices of each monomer which together build up a four-helix bundle. In the crystal structure GrpE forms an asymmetric and bent dimer whereby only one GrpE monomer (orange in fig. ) contacts the ATPase domain of DnaK through five major contact sites.
GrpE binds to the ADP bound and nucleotide-free states of DnaK with high affinity (Kd
= 1 nM). Addition of ATP leads to the dissociation of the complex. As described above, the superposition of the structure of the nucleotidefree ATPase domain of DnaK in complex with GrpE with the structure of the ADP and Pi
bound ATPase domain of Hsc70 [45
] shows that subdomain IIB in DnaK is rotated outwards by 14°, thereby opening up the nucleotide binding cleft. This rotation displaces residues Ser274, Lys270 and Glu267 of DnaK by 2–3 Å. The corresponding amino acids in Hsc70 are involved in the coordination of the adenine and the ribose rings of the bound ADP. Furthermore, residues which constitute the hydrophobic patch and the upper salt bridge (E264-R56) interact with GrpE [60
] (fig. ).
The mechanism by which GrpE accelerates the nucleotide release thus seems to rely on an active opening of the nucleotide binding cleft. GrpE furthermore stabilizes the open conformation of the nucleotide binding pocket, which facilitates the rapid binding of ATP to the nucleotide-free state of DnaK [47
]. In the presence of ATP GrpE thereby regulates the lifetime of the DnaK-substrate complex [63
Bag proteins form a heterogeneous family of multidomain proteins that share the Bag domain frequently located at their C-termini [64
]. This domain is essential and sufficient for stimulation of nucleotide exchange by the mammalian homologs Hsc70 and Hsp70. The nucleotide exchange activity of Bag proteins was first described for the Bag-1 homolog by Höhfeld and co-workers [65
]. Recent kinetic measurements using fluorescent labeled nucleotides showed that Bag-1 stimulates dissociation of ADP from Hsc70 and Hsp70 by up to 100-fold in the absence of inorganic phosphate and by 600-fold in its presence [47
]. Interestingly, Bag-1 does not stimulate the dissociation of ATP and is strongly affected by Pi
, which increased the apparent Kd
by approx. 17-fold. This is in contrast to the interaction of GrpE with DnaK, which also stimulates ATP dissociation and is not affected by Pi
. These observations suggest that the mechanisms by which GrpE and Bag-1 stimulate nucleotide release are different and that Bag-1 plays a more passive role than GrpE. Bag-1 may only bind and stabilize the open conformation of the ATPase domain of Hsc70 during the intrinsic fluctuations between the open and closed states.
The mammalian Bag domains form a three-helix bundle [61
]. It associates as a monomer with the subdomains IB and IIB of the Hsc70 ATPase, through electrostatic interactions involving the conserved residues Glu212, Asp222, Arg237 and Gln245 in Bag-1 (fig. ). Both exchange factors, GrpE and Bag-1, cause the same 14° outwards rotation of subdomain IIB through a hinge.
In addition to GrpE and the Bag proteins other proteins have recently been found to act as nucleotide exchange factors for Hsp70 proteins. The yeast protein Sls1/Sil1, its mammalian homolog Bap and surprisingly the yeast Hsp70-related Hsp170 Lhs1 function as specific exchange factors for the endoplasmatic reticulum resident Hsp70 protein Bip [67
]. Nucleotide exchange function for BiP seems to be essential since double deletion of Lhs1 and Sls1/Sil1 is lethal [71
]. The yeast Fes1 and its mammalian homolog HspBP1 are nucleotide exchange factors for the cytosolic Hsc70 [72
]. In the mammalian cytosol, therefore, several exchange factors coexist, including Bag proteins with currently six distinct family members, one of which exists in four variants, and HspBP1. The physiological role of this diversity is not yet elucidated (see below).