ClpB N-terminal domain plays a regulatory role in protein disaggregation

Abstract

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

“…The conserved NTDs bind hydrophobic regions of substrates and are proposed to direct polypeptides to the NBD pore loops as well as enhance cooperative substrate binding required for amyloid disaggregation 15,29 . In our Hsp104 map the NTDs interact together, forming a broad, open-ended funnel that mirrors the helical arrangement of the NBD rings (Figure 4a).…”

“…Modeling the P2-P4 NTDs was facilitated by localization of the well-defined NTD-NBD1 connecting density as well as regions that could be attributed to the larger A1 and A6 helices that make up the hydrophobic substrate-binding cleft 15 (Figure 4b). Based on our model only the P3 NTD appears to be positioned with its substrate-binding surface facing towards the channel, while the P2 and P4 NTDs each appear to be in different orientations.…”

“…Hsp104 and ClpB contain a mobile N-terminal domain (NTD) implicated in substrate engagement 15,16 , two evolutionarily distinct AAA+ nucleotide binding domains (NBD1 and NBD2) that mediate oligomeric interactions and power polypeptide threading, and a middle domain (MD) that is required for disaggregation and interaction with Hsp70 (Figure 1a) 17–21 . Hsp104 also contains a 38-residue C-terminal domain (CTD) not found in ClpB that is implicated in substrate binding 22 , cochaperone interactions 23 , and is critical for hexamerization 24 .…”

Isotope-labeling strategies for solution NMR studies of macromolecular assemblies

“…Recent work from Rosenzweig et al on the substrate recognition of the 580-kDa hexameric ClpB chaperone demonstrates the dramatic spectral improvement segmental labeling can offer [59 ] (Figure 2c). The Nterminal domain (NTD, 16 kDa) of the ClpB monomer (97 kDa) was expressed as an intein-fusion, with methylgroup specific isotope-labeling, whereas the remainder of ClpB was fully deuterated.…”

Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes

“…Atomic-level information about the formation and dynamics of multi-chaperone, substrate-bound complexes comprises another challenge in the field. Recent NMR work from the Kay group characterized the interaction with client proteins and the 580 kDa Hsp100 (ClpB) hexamer (Rosenzweig et al 2015), but many interesting multi-chaperone or chaperone-substrate complexes remain to be characterized.…”