Asymmetric Interactions of ATP with the AAA+ ClpX6 Unfoldase: Allosteric Control of a Protein Machine

Hersch, Greg L.; Burton, Randall E.; Bolon, Daniel N.; Baker, Tania A.; Sauer, Robert T.

doi:10.1016/j.cell.2005.05.024

Cited by 162 publications

(194 citation statements)

References 42 publications

Supporting

Mentioning

163

Contrasting

Unclassified

Order By: Relevance

“…Studies on a related ATPase ClpX demonstrated complex allosteric linkages between multiple ATP-binding sites resulting in positive as well as negative cooperativity in ATP binding. 22 As it is not known which binding mechanism applies to nucleotide interactions with 12 sites in the hexameric ClpB, we restrict our analysis to observing the shape of binding isotherms to obtain information on the effective nucleotide-binding capability of ClpB.…”

Section: Resultsmentioning

confidence: 99%

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Nagy

et al. 2009

Protein Science

View full text Add to dashboard Cite

Hexameric AAA1 ATPases induce conformational changes in a variety of macromolecules. AAA1 structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA1 sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA1 ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA1 chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's c-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA1 ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

show abstract

Section: Resultsmentioning

confidence: 99%

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Nagy

et al. 2009

Protein Science

View full text Add to dashboard Cite

show abstract

“…The 2 structures available now illuminate the transition from the ADP-to the apo-state. The ATP-loaded hexamer might look roughly similar as the nucleotide-free, but is presumably not 6-fold symmetric anymore, because it is unlikely that 6 ATP bind simultaneously and ATPs are bound with different affinities (24)(25)(26). Furthermore, the nucleotide induced subdomain closure within the AAA moiety would lead to a more compact, closed appearance.…”

Section: Discussionmentioning

confidence: 99%

“…Many other AAAϩ proteins have been crystallized only in the monomeric state and 6-fold-symmetric rings have been modeled frequently (22,23). Recent biochemical data, however, have demonstrated that at least in some nucleotide states the symmetry has to be lower, e.g., only maximally 4 ATP molecules bind to 1 hexameric ClpX or PAN or HslU particle (24)(25)(26).…”

mentioning

confidence: 99%

The crystal structure of apo -FtsH reveals domain movements necessary for substrate unfolding and translocation

Bieniossek

Niederhauser

Baumann

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA؉ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 Å resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo-and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA؉ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.AAAϩ protease ͉ conformational rearrangement ͉ pore residue ͉ FtsH ͉ metalloprotease T he ATPases associated with various cellular activities (AAAϩ) family comprises a large group of proteins which share a common ATPase module, carrying the Walker A and B and other conserved sequence motifs (1, 2). They function as oligomers and usually form hexa-or heptameric rings, whereas in some cases oligomerization is ATP-dependent. AAAϩ proteins are present in all kingdoms of life and participate in a wide range of different actions such as protein disaggregation, complex remodeling, and protein degradation (reviewed in refs. 3-5). In these processes, the energy of ATP is converted into a mechanical force by large conformational rearrangements. Until now, information on these immense structural changes is sparse; x-ray studies on HslU (6) and p97 (7) revealed only relatively small changes between the conformations of apo, ADP, and ATP state. In contrast, electron microscopic studies of p97 (8) and of the chaperone ClpB (9) displayed very large conformational rearrangements of the AAA domains.The membrane-bound metalloprotease FtsH (10, 11) is a homohexameric complex that carries, contrary to most other AAAϩ proteases, the AAA and proteolytic domain on the same polypeptide chain. The AAA domain bears the Walker A and B motifs necessary for nucleotide binding and hydrolysis; the second region of homology (SRH), which carries conserved arginine residues (''arginine fingers'') for oligomerization and nucleotide hydrolysis; and the conserved FGV pore motif required for substrate recognition and translocation. The protease domain exhibits the zincbinding HEXXH finger print. The N-terminal AAA domain is preceded by 2 transmembrane helices flanking a s...

show abstract

“…5). This mechanism would still work to some extent even if hydrolysis or ADP-ATP exchange does not occur in all six AAA domains simultaneously but rather in a probabilistic manner (14,37), as long as there is space for this domain movement. The lack of coupling between ATP hydrolysis and proteolysis in (⌬tm)FtsH constructs, and hence the inability in degrading 32 , could be explained by the absence of ''elastic springs'' formed by the transmembrane helices that would restrain the free movement of the AAA domains and possibly prevent them from being locked into one intermediate conformation on the reaction pathway.…”

Section: Discussionmentioning

confidence: 99%

“…All these substrates are degraded in an ATP-dependent manner where the energy is used for pulling the proteins out of the membrane and͞or unfolding and translocation. However, FtsH possesses only a weak unfoldase activity (13) and is not able to degrade stable proteins, contrary to other ATP-proteases like ClpXP (14).…”

mentioning

confidence: 99%

The molecular architecture of the metalloprotease FtsH

Bieniossek

Schalch

Bumann

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

151

136

View full text Add to dashboard Cite

The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like 32 and -CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.AAA ͉ protease ͉ protein degradation ͉ x-ray A TP-dependent proteases play crucial roles in protein quality control and regulation (for reviews, see refs. 1 and 2). One of these is FtsH, initially described as a temperature-sensitive and cell-division-defective mutant, which is also called HflB, named after a high-frequency of lysogenization locus of bacteriophage . FtsH is an integral membrane protease found in bacteria, chloroplasts, and mitochondria (reviewed in ref.3) (Fig. 6, which is published as supporting information on the PNAS web site). In bacteria, FtsH malfunction causes severe phenotypes like cell division defects and growth arrest (4, 5) (reviewed in ref.3). Deletion of the human mitochondrial homolog paraplegin, which shares 40% sequence identity with FtsH from Escherichia coli, is responsible for an autosomal recessive form of hereditary spastic paraplegia (6). The N terminus of FtsH contains two transmembrane helices followed by an AAA module (ATPases associated with various cellular activities), including the SRH (second region of homology) (3). The C-terminal part of the polypeptide chain bears the HEXXH motif that is characteristic for Zn-dependent metalloproteases, where the two histidines coordinate to the zinc ion and the glutamate serves as a catalytic base. In bacteria, the AAA and protease domains are located on the cytosolic side of the membrane. Of the five ATP-dependent proteases in E. coli, HslVU, Lon, ClpXP, ClpAP, and FtsH, the last is the only one that is essential and universally conserved in bacteria. It degrades membrane proteins like the uncomplexed SecY subunit of translocase (7), the a-subunit of F o F 1 -ATPase (8), and the photosystem in chloroplasts (9), therefore playing an important role in the quality control of membrane proteins. Further targets comprise regulatory soluble proteins such as 32 (10, 11) or -CII transcriptional activator protein (12). All these substrates are degraded in an ATP-dependent manner where the energy is used for pulling the proteins out of the membran...

show abstract

Asymmetric Interactions of ATP with the AAA+ ClpX6 Unfoldase: Allosteric Control of a Protein Machine

Cited by 162 publications

References 42 publications

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

The crystal structure of apo -FtsH reveals domain movements necessary for substrate unfolding and translocation

The molecular architecture of the metalloprotease FtsH

Contact Info

Product

Resources

About