Roles of the N-domains of the ClpA Unfoldase in Binding Substrate Proteins and in Stable Complex Formation with the ClpP Protease

Hinnerwisch, Jörg; Reid, Brian G.; Fenton, Wayne A.; Horwich, Arthur L.

doi:10.1074/jbc.m507879200

Cited by 31 publications

(29 citation statements)

References 50 publications

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“…Overall, we surmise that the I domain is required to stabilize unfolded SP conformations to assist periodic mechanical pulling by the ClpY loops. These results are in accord with experimental studies (39)(40)(41), which suggest that the N domains of ClpA and ClpX stabilize unfolded conformations of substrate proteins and facilitate effective translocation.…”

Section: Moderate Transient Interaction Of the Sp With The I Domain Osupporting

confidence: 81%

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Kravats

Jayasinghe²,

Stan³

2011

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

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Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarsegrained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.AAA+ protease | molecular machine | protein translocation I ntracellular regulatory mechanisms for selective destruction of proteins and disassembly of protein aggregates are critical for the maintenance of vital cellular functions (1). Clp macromolecular machines, found in all domains of life from prokaryotes to multicellular eukaryotes (2), perform such protein quality control using powerful ATPase components (3-6) that effect protein unfolding and translocation through narrow pores. Clp (caseinolytic protease) ATPases, are members of the AAA+ (ATPases associated with diverse cellular activities) superfamily of proteins (7, 8) responsible for a variety of functions including intracellular transport, DNA replication and repair, and transcription regulation (9-11). The best understood Clp ATPases are ClpA; ClpX and ClpY (HslU) (12), which enable protein degradation by associating with peptidase subunits ClpP and ClpQ (HslV); and ClpB, which promotes disaggregation (13-15).Functional forms of Clp ATPases are homohexameric assemblies that form in the presence of ATP (16). Monomers include one (ClpX, ClpY) or two (ClpA, ClpB) highly conserved nucleotide binding domains (17), referred to as AAA domains (7,18). Crystal structures (19-21) and oligomeric models (22, 23) based on electron microscopy images (24, 25) reveal the presence of a central channel with a diameter of approximately 9-15 Å at the narrowest point. The channel is occupied by diaphragm-forming loops (one per AAA domain), which contain a conserved motif consisting of an aromatic-hydrophobic dipeptide flanked by glycine amino acids. These loops are implicated in the ti...

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Section: Moderate Transient Interaction Of the Sp With The I Domain Osupporting

confidence: 81%

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Kravats

Jayasinghe²,

Stan³

2011

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

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“…Binding of ClpP to ClpA almost doubles (ϩ84%) the ATPase activity of the chaperone (Fig. 1e, gray) as shown recently by Hinnerwisch et al (29). ClpAE286A is stimulated to the same extent (ϩ85%), whereas ATP hydrolysis of ClpAE565E is not accelerated when bound to ClpP (Fig.…”

Section: Activity Of the Second But Not Of The First Atpase Domain Ofsupporting

confidence: 51%

“…For example, binding of ClpP stimulates ATP hydrolysis, whereas binding of ClpS on the other hand inhibits the overall ATPase activity (14,29). By analyzing how the two variants are affected, i.e.…”

Section: Discussionmentioning

confidence: 99%

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

Kress

Mutschler²,

Weber‐Ban

2009

Journal of Biological Chemistry

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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...

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“…This threading mechanism is similar to that of the structurally homologous ClpA ATPase, which lacks an M-domain and together with ClpP degrades ssrA-tagged proteins (28)(29)(30). Hsp104 neither associates with ClpP nor degrades proteins but instead releases the unfolded polypeptide from the distal end, which then refolds spontaneously or with assistance from Hsp70/ 40, depending on the substrate.…”

mentioning

confidence: 87%

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

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

104

111

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Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the speciesspecific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.ClpB | DnaK | Hsp100 | molecular chaperone

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Roles of the N-domains of the ClpA Unfoldase in Binding Substrate Proteins and in Stable Complex Formation with the ClpP Protease

Cited by 31 publications

References 50 publications

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

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

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

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