Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent

Park, Eunyong; Lee, Jung Wook; Eom, Soo Hyun; Seol, Jae Hong; Chung, Chin Ha

doi:10.1074/jbc.m805411200

Cited by 15 publications

(24 citation statements)

References 37 publications

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“…Also, in an analysis by real-time monitored surface plasmon resonance, it has been demonstrated that ClpY increases fivefold in binding affinity toward ClpQ while it is associated with MBP-SulA (1). However, deletion of the Thr active site in ClpQ also increases the binding affinity of its subunits with ClpY(s) (25). Using a phage 22 Arc repressor, an artificial substrate, the bound ATP in hexameric ClpY(s) is required for substrate recognition (3).…”

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confidence: 99%

Characterization of the Escherichia coli ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System

Lien¹,

Shy²,

Peng³

et al. 2009

J Bacteriol

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In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.

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confidence: 99%

Characterization of the Escherichia coli ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System

Lien¹,

Shy²,

Peng³

et al. 2009

J Bacteriol

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“…Although HslV has 12 identical active sites, it is not clear whether all of 12 active sites are necessary for efficient substrate degradation or only some of them are sufficient. Previously, we were able to produce mixed HslV dodecamers containing both mutant (T1⌬) and wild-type subunits within the same complex in different ratios, and these dodecamers provided us a hint that only half of the HslV active sites might be sufficient for the full proteolytic activity (17). However, the evidence remains inconclusive because the T1⌬ mutant subunit causes an unexpected, dramatic increase in the affinity between HslV and HslU, which could compensate for the loss of catalytic activity.…”

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confidence: 96%

“…Assuming that the inhibitors mimic the binding of a substrate to the active site, these findings provide a mechanism for the maintenance of stable HslVU complexes when HslVU is engaged in substrate degradation. In addition, deletion of the Thr 1 residues (T1⌬) also causes a dramatic increase in the HslV-HslU interaction even in the absence of ATP (17). Thus, it is clear that the Thr 1 active sites are involved in the tight interaction of HslV with HslU, in addition to the catalytic role in peptide bond cleavage.…”

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confidence: 97%

“…Recently, we have shown that proteasome inhibitors, such as lactacystin or 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucylleucyl-leucyl-vinylsulfone (NLVS) bind to HslV in the presence of HslU and ATP and that binding of these inhibitors to the Thr 1 residues dramatically increases the interaction between HslV and HslU (17). Significantly, the stability of inhibitorbound HslVU complexes is no longer influenced by the presence or absence of ATP.…”

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confidence: 99%

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HslVU ATP-dependent Protease Utilizes Maximally Six among Twelve Threonine Active Sites during Proteolysis

Lee

Park

Jeong

et al. 2009

Journal of Biological Chemistry

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HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome ␤-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 ␤-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only ϳ6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to ϳ6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.ATP-dependent proteases are cellular machines that play essential roles in the controlled turnover of regulatory proteins and the clearance of damaged proteins. They harness chemical energy from ATP hydrolysis, converting it into mechanical force to unfold protein substrates and translocate them into a proteolytic chamber for degradation. These chambers of ATPdependent proteases sequester proteolytic active sites from the cytosol, thus preventing uncontrolled access of cytosolic proteins to the active sites.

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“…In this regard, a detailed understanding of the mechanism of substrate proteolysis and the general role of dynamics in enzyme function could be important for the design of effective pharmaceuticals. Here, we focus on the Haemophilus influenzae HslV protease because high-resolution X-ray structures are available (9,11,12) as well as a significant body of biophysical and biochemical data (14)(15)(16). This protease has an aggregate Significance Molecular machines, such as the proteasome in eukaryotes and archaea as well as the HslU-HslV complex in bacteria, play critical roles in maintaining cellular homeostasis.…”

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confidence: 99%

Tracing an allosteric pathway regulating the activity of the HslV protease

Shi

Kay

2014

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

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Significance Molecular machines, such as the proteasome in eukaryotes and archaea as well as the HslU–HslV complex in bacteria, play critical roles in maintaining cellular homeostasis. Here, we have used methyl-Transverse Relaxation-Optimized Spectroscopy to study the 230 kDa HslV dodecamer that cleaves substrate polypeptides. The initial catalytic step is investigated by measuring the pK a of the terminal amine of catalytic residue T1. Furthermore, we show that single site mutations in key regions, which contact HslU or change on HslU binding, lead to propagated changes in structure along an allosteric pathway, affecting proteolysis rates. NMR relaxation experiments are consistent with a coupling of millisecond timescale dynamics throughout regions of HslV that link HslU and substrate binding with catalysis.

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Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent

Cited by 15 publications

References 37 publications

Characterization of the Escherichia coli ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System

Characterization of the Escherichia coli ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System

HslVU ATP-dependent Protease Utilizes Maximally Six among Twelve Threonine Active Sites during Proteolysis

Tracing an allosteric pathway regulating the activity of the HslV protease

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