Analysis of the Cooperative ATPase Cycle of the AAA+ Chaperone ClpB from Thermus thermophilus by Using Ordered Heterohexamers with an Alternating Subunit Arrangement

Abstract

Background: Ring-shaped ClpB hexamer hydrolyzes ATP and reactivates protein aggregates. Results: Intercalation of ATPase defective subunits into the hexamer every other subunit hampered its ATPase and disaggregation activities. Conclusion: ClpB cooperatively hydrolyzes ATP, and this cooperativity is crucial for protein disaggregation. Significance: This study presents a new method to study the cooperativity of homo-oligomeric proteins and provides insights into the common mechanism of AAAϩ ATPases.

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem

“…Interestingly, ATP binding to the NBD1 also inhibited ATP hydrolysis at neighboring NBD1, indicating that the same event, i.e., ATP binding to NBD1, oppositely regulates the activity of neighbor NBDs located in the same or in opposite rings [99]. In contrast to the aforementioned activation found for Hsp104/ClpB Eco hybrids, incorporation of double Walker B mutant subunits into wt ClpB Tth hexamers reduced the ATPase activity [100], suggesting again that the regulatory circuits might not be identical in these disaggregases and highlighting the importance of allosteric communication to coordinate their ATPase activity.…”

“…This allosteric signal generated upon nucleotide binding to the NBD2 regulates nucleotide binding to the NBD1 of Hsp104 and ClpB Eco [99,102]. In contrast, ClpB Tth lacks this signal as both NBDs showed similar affinities for nucleotides and each of the ClpB Tth NBD ring bound nucleotide non-cooperatively [80,100], reflecting differences in the allosteric communication of these disaggregases.…”

“…A recent work, using heterohexamers with an alternating subunit arrangement formed by the oligomerization of crosslinked dimers that combine subunits with different mutations, confirms coupling of the ATPase activities of both rings and suggests that inter-ring stimulation needs the presence of Arg fingers sensing the ATP state of adjacent subunits within the same ring [100]. This means that coordination of the ATPase activities of both rings is regulated by intra-ring communications.…”

“…The characterization of these mutants has allowed the analysis of the effect of nucleotide binding or hydrolysis in one ring on the activity of the other one. Additionally, intermonomeric communication has been also studied using hybrid hexamers composed of active and inactive subunits containing the aforementioned mutations [67,68,86,88,[97][98][99][100].…”

“…The NBD1 and NBD2 of ClpB/Hsp104 contain two and one conserved arginine residues, respectively, contacting the bound nucleotide ( Figure 3) [38,83]. The role of these conserved residues in the regulation of ATP binding and hydrolysis has been studied for ClpB Tth [89,100,103,106,108]. Arginine fingers are essential for the communication between adjacent NBDs, as mutation of the three conserved arginines (R322, R323 and R747) in full-length ClpB Tth reduced its ATP hydrolysis and threading activity, barely affecting to NBD affinity for nucleotides.…”