The amino‐terminal domain of Mycobacterium tuberculosis ClpB protein plays a crucial role in its substrate disaggregation activity

Abstract

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.

Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain

“…Consistent with the autoinhibitory role of the NTD, ΔNClpB is an activated ClpB variant that has tilted MDs and elevated basal ATP hydrolysis rate, and shows high disaggregation activity toward heat-induced aggregates of G6PDH and firefly luciferase. The increased basal ATP activity exhibited by ΔNClpB in comparison to the full-length variant is in good agreement with multiple preceding studies 17 , 19 , 20 , 31 − 36 and implies that this truncated mutant of ClpB is more wasteful in terms of ATP consumption in the absence of bound substrate–proteins. Our data show unaltered yield and a faster rate of disaggregation following the NTD removal.…”

Single-molecule spectroscopy reveals dynamic allostery mediated by the substrate-binding domain of a AAA+ machine

“…Based on our results, ΔNClpB is an activated ClpB variant that has tilted MDs, elevated basal ATP hydrolysis rate and high disaggregation activity towards heat-induced aggregates of G6PDH and firefly luciferase. The increased basal ATP activity exhibited by ΔNClpB in comparison to the full-length variant is in good agreement with multiple preceding studies (10,19,20,(28)(29)(30)(31)(32)(33), and implies that this truncated mutant of ClpB is more wasteful in terms of ATP consumption in the absence of bound substrate-proteins. Our data show unaltered yield and faster rate of disaggregation following the NTD removal.…”

“…However, accumulating experimental evidence suggests that their role extends beyond substrate-protein binding. Numerous studies reported increased ATP hydrolysis rate by the NTD-truncated mutant ΔNClpB in the absence of protein substrates, compared to the fulllength ClpB, indicating that the NTDs may inhibit futile ATP hydrolysis through yet uncharacterized communication pathways (10,19,20,(28)(29)(30)(31)(32)(33). Indeed, potentially relevant interactions of the NTD with the MD and NBDs in solution were recently detected by X-ray footprinting in Hsp104 (34).…”

Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation

“…Recent reports illustrate a role of the NTD in regulating ClpB activity. A cryo EM model of ATP-bound E. coli ClpB shows a single NTD that is located on top of the central translocation channel, sealing the pore entrance ( Figure 3 ) ( Deville et al, 2017 ; Tripathi et al, 2018 ). The substrate binding groove of the NTD remains accessible in this state providing a pathway for substrate-induced ClpB activation.…”