![]() ![]() The invariant D of the J-motif interacts with the NBD and leads to a destabilization of the domain-domain interface of the NBD, resulting in ATP hydrolysis. The ATPase activity of BiP can be stimulated via interaction with J-domain containing proteins through their defining conserved H-P-D or J-motif ( Pobre, Poet, and Hendershot 2019). Transition between the two states is regulated allosterically by the nucleotide bound state of the NBD. Hsp70 proteins occupy two main conformations, an open or docked state (ATP bound), or a closed or undocked state (ADP bound). The nucleotide binding pocket contains the adenine nucleotide sensing residue T 37, which interacts with the γ-phosphate of the nucleotide, distinguishing ADP and ATP ( Wei, Gaut, and Hendershot 1995). The NBD of BiP is a large globular domain organized into two large lobes with a deep cleft containing the nucleotide binding pocket between them ( Pobre, Poet, and Hendershot 2019). FICD AMPylates BiP as a monomer and de-AMPylates BiP as a dimer (5) (AMP shown on the structure in red) (PDB: 6I7K, SO4P, 6I7G, respectively). BiP sequestering can be achieved through modification, specifically AMPylation via FICD. Nucleotide exchange is facilitated by a NEF (Sil1) (4), exchanging ADP for ATP, which causes BiP to unclamp from the substrate and occupy the ATP- bound open conformation once more (5) (PDB: 3QML). Upon stimulation, BiP hydrolyzes ATP to ADP and the SBD clamps onto the substrate (3), undocking from the NBD for more dynamic movement (PDB: 5E85). Here, ERdj6 brings substrate (red) bound in the TPR domain (orange) to pass to BiP (2), then stimulates the ATPase activity of BiP via the J-domain (grey) (PDB: 2Y4K). Substrate may be bound directly or transferred to BiP via a co-factor. In the ATP-bound state, BiP is in the open conformation (1), with both the SBD (purple) and NBD (green) domains docked together. Altogether, the Hsp70 and carbohydrate-dependent chaperone networks of the ER assist a broad range of clients that pass through the secretory pathway to help maintain cellular homeostasis and these chaperone networks are the focus of this chapter.Īn overview of the BiP substrate binding cycle. The carbohydrate-dependent chaperone system binds to the N-glycan modification based on their dynamic composition to aid in folding and quality control of the cargo they are attached to ( Hebert et al. Proteins that traverse the secretory pathway are frequently modified by N-linked glycans, and a chaperone system has evolved to utilize these glycans. In addition to assisting in protein folding, BiP aids in protein translocation, as well as the preparation and targeting of terminal misfolded for degradation. In the endoplasmic reticulum (ER) where proteins targeted to the eukaryotic secretory pathway mature the Hsp70 family member is BiP (Binding-immunoglobulin Protein or Grp78) ( Pobre, Poet, and Hendershot 2019). Hsp70 is a well conserved classical family of chaperones that is ubiquitously expressed throughout the cell and performs diverse roles ( Balchin, Hayer-Hartl, and Hartl 2020). Prolonged interactions with chaperones can also play a central role in quality control decisions that supports the targeting of irreparable misfolded proteins for degradation. By transiently interacting with the maturing nascent chains molecular chaperones increase the folding efficiency in the suboptimal folding environment of the cell. The folding of more complex proteins however, frequently requires assistance from cellular folding factors. Here, we compare and contrast the properties, mechanisms of action and functions of these different chaperones systems that work in parallel, as well as together, to assist a large variety of substrates that traverse the eukaryotic secretory pathway.Īnfinsen’s seminal studies demonstrated that the primary sequence of the protein determines the three-dimensional structure of the folded protein and that small proteins can fold in isolation in the test tube ( Anfinsen 1973). In contrast, the carbohydrate-dependent chaperone system involving the membrane protein calnexin and its soluble paralogue calreticulin recognize a specific glycoform of an exposed hydrophilic protein modification for which the composition is controlled by a series of glycosidases and transferases. ![]() The ER Hsp70 chaperone, BiP, supports adenine nucleotide-regulated binding to non-native proteins that possess exposed hydrophobic regions. There are two major molecular chaperone families in the endoplasmic reticulum (ER) that assist proteins in reaching their native structure and evaluating the fidelity of the maturation process. Chaperones also aid in quality control decisions as persistent chaperone binding can help to sort terminal misfolded proteins for degradation. Molecular chaperones assist the folding of nascent chains in the cell. ![]()
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