Grp78/BiP ist the only ER-resident member of the huge and highly conserved HSP70 family of molecular chaperones. Grp78/BiP primarily exist as a homodimer which is subjected to post-translational modifications 49. Nevertheless, higher oligomeric states have also been reported 52. All HSP70s share a common structure consisting of two conserved functional domains: (i) an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate binding domain (SBD) 38-40 connected by a short hydrophobic inter-domain linker. This linker has been proposed to mediate homo-oligomerization of Grp78/BiP (Figure 1). The NBD (also termed ATP-binding domain, ABD) is composed of four subdomains (IA, IB, IIA, IIB) surrounding the ATPase-binding pocket (Figure 2) 30, 96. The NBD binds and hydrolizes ATP while the SBD binds to unfolded polypeptides 38–40. The SBD is composed of an N-terminal two-layered twisted β-sheet (SBDβ) and a C-terminal α-helical subdomain (SBDα) providing a flexible lid of SBDβ 97-99.
Central to the chaperone function of Grp78/BiP is the transition between the ATP-bound open and ADP-bound closed conformation of the SBD, driven by ATP via allosteric linking of the NBD and SBD 100, 101. ATP binding to the NBD alters the substrate affinity to the SBD and substrate binding to stimulate ATP hydrolysis 102. In the nucleotide-free (apo) and ADP-bound states (also referred to as domain-undocked conformation), only weak interactions take place between the two functional domains (Figure 3) 102-104. The substrate binding characteristics of this state are labelled by high affinity and slow binding/release rates 102, 105-107. In the ATP-bound (domain-docked) state, NBD and SBD are tightly coupled revealing a conformation with low ATPase activity and affinity as well as accelerated binding/release rates for the substrate 102, 106, 108, 109. Recruitment of the substrate preserves the domain-undocked conformation, now characterized by high ATPase activity, fast binding/release rates and low substrate affinity 102, 107. It is noteworthy that this allosteric interaction plays a pivotal role in facilitating an effective chaperone activity 100. Most recently, the group of Qinglian Liu solved the structures of the isolated SBD in Grp78/BiP. Their data led to the assumption of a highly conserved opening of the substrate binding pocket in Grp78/BiP induced by ATP binding 31. On the molecular level, it became obvious that the hydrophobic inter-domain linker associates with the NBD upon ATP binding while NBD and SBDβ interact with the SBDα lid which is disassembled from SBDβ and thus able to associate with the NBD. As a consequence, SBDβ opens up and allows for the recruitment of the substrate polypeptide 31. Analysis of methyl NMR spectra of an ATPase-deficient variant of Grp78/BiP and its NBD revealed a marked discrepancy in the distribution of the domain-docked and ‑undocked conformations for different ligand-bound states. ATP-bound Grp78/BiP has been identified to exist in solution either in a domain-docked conformation and, to a minor degree, an ADP-like domain-undocked conformation 31. It is of note that ATP binding obviously does not play a role in adopting the domain-docked conformation while AMPylation of the SBD has been shown to stabilize this conformational state in the presence of adenine nucleotides 31.
Re-opening of the SBDα lid and release of the substrate are achieved by nucleotide exchange factors (NEFs) that participate in ADP/ATP exchange 110, 111. HSP70s including Grp78/BiP are assisted not only by NEFs but also by various co-chaperones called J-domain proteins (HSP40s) 112. Members of the HSP40/DNAJ family stimulate the intrinsic ATPase activity of HSP70s and coordinate it with substrate binding by interacting with the HSP70 ATPase domain through their J-domain 113. Growing evidence suggests that AMPylation facilitates rapid substrate release and blocks enhancement of ATP hydrolysis by J-domain proteins 114. Additionally, the allosteric model proposed by Steffen Preissler and David Ron illustrates increased preservation of the hydrophobic inter-domain linker from proteolysis and linker-mediated Grp78/BiP homo-oligomerization as occurring in AMPylated Grp78/BiP in the presence of either ADP or ATP 114. For more detailed information on ATP-induced conformational re-arrangements in HSP70s see HSP70 Scientific Resource Guide and appropriate literature, for instance 30-32, 115.