Assembly Pathway of an AAA+ Protein: Tracking ClpA and ClpAP Complex Formation in Real Time

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.

Section: Volume 286 • Number 35 • September 2 2011mentioning

confidence: 99%

Stalled Proteasomes Are Directly Relieved by P97 Recruitment

Isakov

Stanhill

2011

“…ATP binding to the first ATPase domain of ClpA triggers the assembly into a hexameric ring structure, which is the prerequisite for activity and for binding to the protease ClpP (10,21). Therefore, we investigated the assembly state of all ClpA variants and their ability to associate with the protease ClpP.…”

Section: Atpase Activity Of the First And The Second Aaa Module Canmentioning

confidence: 99%

“…ClpP, ClpA, and all ClpA variants were purified as described previously (21). ClpS was purified as established by Dougan et al (13).…”

mentioning

confidence: 99%

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Kress

Mutschler²,

Weber‐Ban

2009

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ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.The chaperone ClpA is a member of the AAA ϩ protein family (ATPase-associated with various cellular activities) and catalyzes the energy-dependent degradation of proteins through interaction with the protease ClpP in Escherichia coli (1-3). Like many other AAA proteins (4), ClpA oligomerizes into a ring structure, shaping a central pore through which substrate proteins are routed into the proteolytic core ClpP. This process involves unfolding and translocation of the substrate protein and requires the consumption of ATP. AAA proteins can be grouped into class I (two ATPase domains) and class II (one ATPase domain) ATPases. The fact that some ATPases have only one AAA module whereas others seem to require two ATPase domains stimulated researchers to investigate the roles and interdependence of the two ATP-binding modules in class I AAA proteins like Hsp104 and ClpB (5-8). ClpA also features two ATPase domains, termed D1 and D2. They are highly homologous, but differences in the amino acid sequence around the conserved regions in D1 and D2 suggested that they might have a somewhat different function (9). As was shown for several other class I members, ClpA assembles into its oligomeric state only upon binding of nucleotide (10). Indeed, substituting the invariant lysine in the Walker A motif demonstrated that nucleotide binding to D1 triggers ClpA hexamerization, whereas ATP turnover is mainly catalyzed by D2 (11, 12). However, mutations in the Walker A motif also abolish or drastically decrease the affinity for ATP, making it impossible to distinguish between effects due to ATP binding and those due to ATP hydrolysis.To study the role of ATP hydrolysis in both ATPase domains uncoupled from nucleotide binding events, we designed ClpA Walker B variants that lack the ability to hydrolyze ATP in either D1 (ClpAE286A), D2 (ClpAE565A), or both domains (ClpAE286A/E565A) but still bind ATP in bot...

“…We collected a dataset of 13 time traces at stoichiometric and nonstoichiometric mixing ratios and fitted them globally to different possible association pathways. Such global fitting procedures of different datasets have been shown to be adequate for elucidating sophisticated binding pathways (28,29). Surprisingly, the minimal association mechanism that describes the data sufficiently is a simplified, quasi-irreversible two-step model (Scheme 1) that involves a conformational change of PezT (T) after binding to an empty PezA binding site (A i ),…”

Section: Pezt Remains Inactive After Inhibition Of Pezat Expression Imentioning

confidence: 99%

Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

Mutschler

Reinstein

Meinhart

2010

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The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation. Toxin-antitoxin (TA)2 systems were initially discovered on prokaryotic low-copy number plasmids, where they ensure stable inheritance in a growing population. By a postsegregational killing mechanism these systems trigger cell death of offspring that have lost the plasmid and were thus also designated as addiction or suicide modules. Most of those TA systems are transcribed from a single bicistronic operon, where the first gene product (antitoxin) inhibits the cytotoxic function upon binding to the product of the succeeding gene (toxin) (1). Addiction to the TA locus is achieved by differential proteolytic stabilities of the two proteins because antitoxic proteins are metabolically less stable and prone to faster degradation by intracellular proteases when compared with their cognate toxins. Hence, loss of the TA-loci results in a faster reduction of the pool of antitoxic proteins than that of toxins due to lack of de novo synthesis, which eventually confronts the host with the freed and corruptive activity of the toxin.In addition to plasmid-encoded TA systems, similar modules were also discovered to be encoded from chromosomes of freeliving bacteria (2). However, the function of these chromosomally encoded TA systems is currently under debate (3, 4) and different functions such as growth control (5), stabilization of mobile genetic elements (6, 7), or programmed cell death (8) have been suggested.TA systems of the ⑀/ family are encoded on various plasmids or chromosomes of Gram-positive pathogens like Streptococcus pyogenes or Streptococcus pneumoniae as well as Gramnegative such as Fusobacterium nucleatum (9 -12). The related -toxins are ϳ30-kDa monomeric proteins owning a monophosphate kinase-fold and are thought to phosphorylate a still enigmatic substrate (13). In addition to the toxin-inactivating function of the antitoxin ⑀, various homologues harbor a transcriptional repressor domain that regulates the operon (11, 14).The PezAT system (pneumococcal epsilon zeta) from S. pneumo...