Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation

Kenniston, Jon A.; Burton, Randall E.; Siddiqui, Samia M.; Baker, Tania A.; Sauer, Robert T.

doi:10.1016/j.jsb.2003.10.023

Cited by 56 publications

(61 citation statements)

References 32 publications

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“…We presume that the less force-dependent step likely involves the translocation of the unfolded chain, because it is not limited by unfolding. Remarkably, this putative translocation barrier imposes an overall speed limit on the translocation of unfolded protein substrates (on the order of Ϸ10 s for Ϸ100-700-residue proteins) for our system (4) and others (6)(7)(8)26); and this limit on the translocation step is invariant with the type of driving force applied (whether ATP, ⌬⌿, or ⌬pH). Understanding the common features shared among these and other translocases will advance our understanding of cellular protein unfolding.…”

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 96%

“…Prior studies indicate that translocation is limited by the local stability near the signal tag (2,8,26). Once this local secondary structure near the signal tag is unfolded, the rest of the protein is thought to denature rapidly (6).…”

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 99%

“…Better engagement with the channel or motor could increase the driving force applied to the substrate such that unfolding is no longer rate-limiting. In fact, when the 12-residue ClpXP degradation tag (26) and 65-residue mitochondrial import presequence (28) are lengthened Ϸ10 residues, the rate of translocation is accelerated Ϸ10-and Ϸ3-fold. Therefore, local unfolding near the signal tag may result in partially unfolded intermediates, but the driving force may have been too high, such that the unfolding of the remaining structure in the protein was not rate-limiting.…”

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 99%

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Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

Thoren¹,

Worden

Yassif

et al. 2009

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

125

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Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal ␤-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.unfolding pathway ͉ transition state structure ͉ mechanical unfolding F olded proteins are Ϸ5-10 kcal⅐mol Ϫ1 more stable than their unfolded states. Therefore, the disassembly and translocation of folded proteins often require a molecular machine and a source of free energy. These ubiquitous multiprotein complexes include soluble degradation machinery, such as the proteasome or the Clp bacterial proteases (1), which unfold and degrade proteins, and some, but not all, membrane-embedded translocase channels, which can unfold and transport proteins across membranes (2). There are general features shared between these soluble and membrane-embedded translocase machines: a narrow central pore first engages the protein substrate on its free end, the substrate is unfolded mechanically, and the unfolded chain is translocated through the narrow pore, allowing it ultimately to either cross a membrane or enter into a proteolytic complex for degradation. Protein unfolding and translocation in these systems are often driven by ATP hydrolysis (1, 2), a membrane potential (⌬⌿) (2, 3), and/or a proton gradient (⌬pH) (4). The molecular mechanism of translocation-coupled unfolding, however, is poorly understood.Prior studies examining the correlation between substrate protein stability and translocation kinetics have produced conflicting results. Some ligand-stabilized substrates translocate inefficiently, because they are too thermodynamically stable (5); however, other substrates show little change in the rate of translocation when destabilized by mutagenesis (6, 7). To resolve these conflicting results, it was proposed that translocation-coupled unfolding (6, 8) depends on the mechanical stability of the local structure adjacent to the signal tag. O...

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Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 96%

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 99%

Section: Does Local Structure Control Translocation-coupled Unfolding?mentioning

confidence: 99%

See 1 more Smart Citation

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

Thoren¹,

Worden

Yassif

et al. 2009

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

125

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“…PezAT Complex Formation Strongly Increases the Thermodynamic Stability of the Two Proteins-Protein susceptibility to degradation depends on their global thermodynamic stability and, if they are substrates for ATP-dependent proteases on the accessibility and local stability of recognition motifs (25)(26)(27). To probe if the observed proteolytic stability of the PezAT complex is correlated with an increased thermodynamic stability, we performed unfolding studies of PezAT and both individual proteins using urea-induced denaturation monitored by the red shift in tryptophan fluorescence and circular dichroism.…”

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

Journal of Biological Chemistry

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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...

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“…We have been interested in understanding the coordination of substrate binding, denaturation, and translocation by the ClpXP protease of Escherichia coli (3)(4)(5)(6). Most substrates for this ATP-dependent protease have unstructured recognition sequences or degradation tags at their N or C terminus (7).…”

mentioning

confidence: 99%

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

Kenniston

Baker

Sauer

2005

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

123

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Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in all cells. AAA؉ ATPases in these proteases bind protein substrates and power their mechanical denaturation and subsequent translocation into a secluded degradation chamber where polypeptide cleavage occurs. Here, we show that model unfolded substrates are engaged rapidly by ClpXP and are then spooled into the degradation chamber at a rate proportional to their length. Degradation and competition studies indicate that ClpXP initially binds native and unfolded substrates similarly. However, stable native substrates then partition between frequent release and infrequent denaturation, with only the latter step resulting in committed degradation. During degradation of a fusion protein with three tandem native domains, partially degraded species with one and two intact domains accumulated. These processed proteins were not bound to the enzyme, showing that release can occur even after translocation and degradation of a substrate have commenced. The release of stable substrates and committed engagement of denatured or unstable native molecules ensures that ClpXP degrades less stable substrates in a population preferentially. This mechanism prevents trapping of the enzyme in futile degradation attempts and ensures that the energy of ATP hydrolysis is used efficiently for protein degradation.ClpP ͉ ClpX ͉ energy-dependent proteolysis ͉ protein unfolding ͉ titin I27 domain M any cellular processes are powered by molecular machines, which convert energy from ATP into mechanical work. The AAAϩ superfamily of ATPases represent an important class of these machines, functioning in vesicle fusion, cargo transport, DNA and RNA unwinding, remodeling of the cytoskeleton, DNA replication, transposition, and targeted protein degradation (1). In known energy-dependent proteases, hexameric AAAϩ ATPase rings stack against a barrel-shaped peptidase, aligning the central pore of the ATPase with a narrow axial portal of the peptidase. The ATPase ring serves as the control and command center for the proteolytic machine. It binds recognition elements in target proteins, denatures native protein substrates, and translocates the unfolded polypeptide into the proteolytic chamber of the peptidase for degradation (see ref. 2 for review).We have been interested in understanding the coordination of substrate binding, denaturation, and translocation by the ClpXP protease of Escherichia coli (3-6). Most substrates for this ATP-dependent protease have unstructured recognition sequences or degradation tags at their N or C terminus (7). For example, adding an ssrA tag to the C terminus of a protein during tmRNA-mediated ribosome rescue makes it a substrate for ClpXP and other E. coli proteases (8-10). The ssrA tag appears to bind to the central pore of the ClpX 6 ATPase (11), where it serves as a grip or handle that allows the enzyme to apply an unfolding force to the native substrate. Peptide-bond cleavage and product release appear to be fast steps...

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Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation

Cited by 56 publications

References 32 publications

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

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

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

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