Most proteins need to fold into a precise structure encoded in their amino acid sequence in order to have proper function. Failure in protein folding can result in dysfunction
and protein aggregation that are toxic to the cell. Protein biogenesis starts with the ribosome synthesizing polypeptide, during which polypeptide chain passes through a narrow ribosomal tunnel and emerges into the cytosol at roughly 35 amino acids long. The chain can start acquiring structures based on the available sequence outside the tunnel. However, since the sequence is not complete, there is risk for misfolding or non-specific interaction with the crowded cytosol. Therefore, the cell has chaperones specialized in assisting the folding and preventing unwanted interaction cotranslationally. Although the presence of cotranslational chaperone is universal, different species arrive at diverse solutions throughout the evolution.
In bacteria, trigger factor (TF) is the major cotranslational chaperone, which forms a hydrophobic cradle right outside the ribosomal tunnel and binds to the nascent chain to prevent misfolding106. In eukaryotic cells, two cotranslational chaperones have been identified: nascent polypeptide associated complex (NAC), and ribosome associated complex (RAC)5. NAC is a heterodimer of NACα and NACβ, forming a small globular domain hovering at the ribosomal tunnel exit anchored by the highly positively charged NACβ N-terminal tail107. NAC is close in stoichiometry to the ribosome and binds to the ribosome at low nanomolar affinity, suggesting that all translating ribosome in the cytosol are bound by NAC82. NAC has been shown to improve the specificity of cotranslational protein targeting to endoplasmic reticulum (ER) through substrate triage with signal recognition particle (SRP) by the UBA domain on NACα107. Both TF and NAC operate passively without energy input.
On the other hand, HSP40/HSP70 is a major class of chaperones conserved throughout the evolution. HSP70 contains two domains: nucleotide binding domain (NBD)
and substrate binding domain (SBD)108. NBD binds to and hydrolyzes ATP to utilize the free energy to assist protein substrate folding. SBD contains a cradle-like subdomain that can bind to a peptide chain roughly 7 amino acids long and another subdomain that forms a long helix acting like a lid closing onto the cradle. The conformation of SBD is controlled by the nucleotide state of NBD: close state with ADP and open state with ATP. The hydrolysis cycle of HSP70 is regulated by HSP40, substrate and nucleotide exchange factor (NEF).
HSP40 is a class of diverse proteins containing the homologous J-domain. The universally conserved HPD tripeptide in the J-domain directly interacts with HSP70 in the junction between NBD and SBD to stimulate its ATP hydrolysis109. Some HSP40 also bind to their substrates and deliver them to HSP70, resulting in a diverse pool of protein substrates chaperoned by HSP40/HSP70 system110. To complete the ATP hydrolysis cycle, NEF binds to HSP70 in its ADP state to accelerate the exchange of ADP to ATP. During the ATP hydrolysis cycle, substrate binding to HSP70-ATP is kinetically fast but thermodynamically weak, vice versa for HSP70-ADP. The binding to HSP70 is believed to favor an unfolded state of substrate, likely due to entropic force exerted by HSP70 binding111. The ATP cycle of HSP70 thus biases the substrate to first unfold from its current structure and then be released in a high free energy unfolded state to refold112. This cycle of unfolding and refolding with ATP free energy input is believed to resolve misfolded states and aggregates and disassemble existing cellular structures for recycling.
Curiously, in eukaryotic cells, there is a ribosome associated HSP40/HSP70 system, RAC, specialized in chaperoning de novo folding of NC. RAC is a heterodimer of DNAJC2 (Zuo1 in yeast) and HSPA14 (Ssz1 in yeast), an unconventional duo of HSP40 and HSP70.
DNAJC2 is the HSP40 and contains the N-terminal domain (NTD), J-domain, Zuotin homology domain (ZHD), a long helical middle domain (MD), 4 helical bundle (4HB) domain and a long C-terminal extension (not present in yeast Zuo1) with two SANT domains of unknown function. HSPA14 is a HSP70 homologue but does not hydrolyze ATP nor has known chaperone activity like a conventional HSP70113. Instead, RAC recruits cytosolic HSP70 like HSPA1A or HSPA8 (Ssb1/2 but not Ssa in yeast) to perform chaperone activity near the ribosomal tunnel exit114,115. RAC binds to the ribosome mainly through DNAJC2, spanning the small (SSU) and the large subunit (LSU) of ribosome116–118. The mammalian RAC (mRAC) is homologous to the yeast RAC and can rescue the deletion of yeast RAC when expressed through a plasmid119. Most of the molecular information has come from yeast RAC and inferred for mRAC based on homology. Based on the recent structures of yeast RAC-ribosome complex, the 4HB domain of Zuo1 interacts with ES12 on the SSU while the ZHD interacts with eL31 on the LSU116. The two ribosome interacting sites are bridged by MD, of which length is conserved and important for function117. The ZHD is connected to the J-domain of Zuo1 placing it close to the ribosomal tunnel exit. The Zuo1 N-terminal domain is attached to the J-domain through a flexible linker and directly interacts with Ssz1 to dimerize120. Ssz1 does not directly interact with the ribosome and may flop around near the ribosomal tunnel exit, as shown by the variable conformations and positions of Ssz1 in the recent structures116. There are studies showing that Ssz1 might be involved in recruiting Ssb through interaction between the two NBD or interacting with the nascent chain through its SBD121,122. However, the exact role of Ssz1 is not known. Despite these recent advances, it is not clear how RAC, especially mammalian one, coordinates the recruitment
of HSP70 to the ribosome, NC binding to the HSP70 and the activation of HSP70 ATP hydrolysis.
In this study, we developed ATP hydrolysis assays to directly measure the activation of HSP70 by RAC. We found that HSP70 binds to RAC alone weakly and does not activate ATP hydrolysis significantly. However, upon RAC association with ribosome, HSP70 is significantly activated by RAC at high affinity. The ribosome-dependent activation of HSP70 by RAC is not affected by the deletion of C-terminal domain of RAC but is perturbed by HSPA14 NBD deletion. We further developed an assay to test cotranslational interaction between HSP70 and NC based on nanoluciferase (NLuc). HSP70 interacts with NC only when stimulated by RAC. The interaction with HSP70 delays the start of folding of NC by keeping NC in a folding-competent unfolded state.