Abstract:ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various comb… Show more
“…Remarkably, this mutant was found to be hyperactive in terms of its ATPase activity (5 folds higher than the WT), though it showed similar disaggregation activity to the WT (Fig. 5c), as also reported by Hayashi et al 82 .Fig. 5Mutations that change M-domain dynamics affect ClpB function.…”
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of ClpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of ClpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of ClpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.
“…Remarkably, this mutant was found to be hyperactive in terms of its ATPase activity (5 folds higher than the WT), though it showed similar disaggregation activity to the WT (Fig. 5c), as also reported by Hayashi et al 82 .Fig. 5Mutations that change M-domain dynamics affect ClpB function.…”
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of ClpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of ClpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of ClpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.
“…Repr-ΔNClpB had slightly decreased (by 10%) ATPase activity compared to ΔNClpB, and its disaggregation activity was reduced by half compared to the non-mutated ΔNClpB (Fig. 6a,b), in good agreement with the previously observed effects of this mutation in the full-length protein (18,51,52). The FRET efficiency histogram of the repr-ΔNClpB was shifted to lower average values in comparison to ΔNClpB.…”
Section: Ntd Removal Does Not Disrupt Communication Between the MD Ansupporting
confidence: 90%
“…Point-mutation E283A within the MD (repr-ΔNClpB, corresponding to E423A in the fulllength ClpB) stabilizes its contacts with NBD1 and with MDs of neighboring subunits (14,15,51,52). Repr-ΔNClpB had slightly decreased (by 10%) ATPase activity compared to ΔNClpB, and its disaggregation activity was reduced by half compared to the non-mutated ΔNClpB (Fig.…”
Section: Ntd Removal Does Not Disrupt Communication Between the MD Anmentioning
ClpB is an ATP-dependent protein disaggregation machine that is activated on demand by co-chaperones and by aggregates caused by heat shock or mutations. The regulation of ClpB's function is critical, since its persistent activation is toxic in vivo. Each ClpB molecule is composed of an auxiliary N-terminal domain (NTD), an essential regulatory middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains that are responsible for ATP-fuelled substrate threading. The NTD is generally thought to serve as a substrate-binding domain, which is commonly considered to be dispensable for ClpB's activity, and is not well-characterized structurally due to its high mobility. Here we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its involvement in novel allosteric interactions. We find that the NTD fluctuates on a microsecond timescale and, unexpectedly, shows little change in conformational dynamics upon binding of a substrate protein. During its fast motion, the NTD makes crucial contacts with the regulatory MD, directly affecting its conformational state and thereby influencing the overall ATPase and unfolding activity of this machine. Moreover, we also show that the NTD mediates signal transduction to the nucleotide-binding domains through conserved residues. The two regulatory pathways revealed here enable the NTD to suppress the MD in the absence of protein substrate, and to limit ATPase and disaggregation activities of ClpB. The use of multiple parallel allosteric pathways involving ultrafast domain motions might be common to AAA+ molecular machines to ensure their fast and reversible activation.
“…We found the ATPase activity of ClpB∆N, the Mtb ClpB variant lacking the NTD, to be higher than that of the full length protein, which could be an indirect effect of changes in the pore shape leading to enhanced nucleotide binding. Further, this study shows that DnaK stimulates the ATPase activity of ClpB, but the effect is only moderate as DnaK homologs are reported to realize their full stimulatory potential in its aggregate bound state . In addition, we also show that unstructured polypeptide substrates interact with the mycobacterial ClpB and differentially stimulate its ATPase activity.…”
Mycobacterium tuberculosis
(
Mtb
) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved molecular chaperone machinery. ClpB, a unique member of the
AAA
+ family of chaperones, is responsible for resolving aggregates in
Mtb
and many other bacterial pathogens.
Mtb
produces two isoforms of ClpB, a full length and an N‐terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N‐terminal domain (
NTD
) of
Mtb
ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of
Mtb
ClpB. We found the
NTD
to be dispensable for oligomerization,
ATP
ase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the
NTD
of
Mtb
ClpB has a hydrophobic groove that contains four hydrophobic residues: L97, L101, F140 and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.
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