Structural determinants stabilizing the axial channel of <scp>ClpP</scp> for substrate translocation

Alexopoulos, John A.; Ahsan, Bilal; Homchaudhuri, Lopamudra; Husain, Nabiha; C, Yi

doi:10.1111/mmi.12356

Cited by 24 publications

(20 citation statements)

References 43 publications

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“…The N-loops of ClpP1 and ClpP2 differ in their length, about 7 residues in ClpP1 versus 17 residues in ClpP2, and do not show structural similarities. The N-loop of ClpP2 is well resolved in the crystal structure, and contains a β-hairpin necessary for efficient substrate translocation [ 17 , 24 , 41 ], while the ClpP1 N-loop residues are resolved neither in the ClpP1P1 nor the ClpP1P2 structure [ 24 , 42 ], indicating a large degree of flexibility. While the N-loops are not resolved in ClpP1 in either structure, the apparent axial pore size of ClpP1 in the inactive ClpP1P1 structure (12 Å) differs substantially from the one of ClpP1 in the active ADEP-bound ClpP1P2 structure (30 Å), indicating that ADEP-binding to ClpP2 allosterically opens the ClpP1 pore [ 24 ].…”

Section: Discussionmentioning

confidence: 99%

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

2015

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Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.

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Section: Discussionmentioning

confidence: 99%

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

2015

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“…Binding of different ADEPs has been shown to activate degradation of unfolded proteins by ClpP (28). ADEPs allosterically induce a change in binding interactions between the N-terminal ␤-hairpin and residues on the surface of ClpP, causing a reorientation of the ␤-hairpins and widening of the axial channel, which presumably allows unfolded polypeptides to pass into the degradation chamber (26,39). In the crystal structures Bz-LL and Z-LL both promoted an open axial channel configuration in ClpP2.…”

Section: Opposite Orientations Of the Activating Dipeptide In Clpp1mentioning

confidence: 99%

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

Kandror

Akopian

et al. 2016

Journal of Biological Chemistry

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The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel ␤-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

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“…Interestingly, most chaperone systems are co-localized to the heat-induced protein aggregates in E. coli (Winkler et al 2010 ). Besides, periplasmic protease DegP, the CpxAR (Cpx) mechanism, involvement of BaeRS (Bae), and the events of Rcs phosphorelays, the phage shock (PSP) response, and the responses generated by ppGpp in response to heat shock become functional (Alexopoulos et al 2013 ; Barchinger and Ades 2013 ; Kumar and Sourjik 2012 ; Morimoto 2012 ; Suzuki et al 2012 ; Majdalani and Gottesman 2005 ; Raivio 2005 ; Rowley et al 2006 ; Ruiz and Silhavy 2005 ; Artsimovitch et al 2004 ; Porankiewicz et al 1999 ).…”

Section: Cell Survival Employing Heat-shock Proteins (Hsps) In Concermentioning

confidence: 99%

Mechanism to control the cell lysis and the cell survival strategy in stationary phase under heat stress

Noor

2015

SpringerPlus

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An array of stress signals triggering the bacterial cellular stress response is well known in Escherichia coli and other bacteria. Heat stress is usually sensed through the misfolded outer membrane porin (OMP) precursors in the periplasm, resulting in the activation of σE (encoded by rpoE), which binds to RNA polymerase to start the transcription of genes required for responding against the heat stress signal. At the elevated temperatures, σE also serves as the transcription factor for σH (the main heat shock sigma factor, encoded by rpoH), which is involved in the expression of several genes whose products deal with the cytoplasmic unfolded proteins. Besides, oxidative stress in form of the reactive oxygen species (ROS) that accumulate due to heat stress, has been found to give rise to viable but non-culturable (VBNC) cells at the early stationary phase, which is in turn lysed by the σE-dependent process. Such lysis of the defective cells may generate nutrients for the remaining population to survive with the capacity of formation of colony forming units (CFUs). σH is also known to regulate the transcription of the major heat shock proteins (HSPs) required for heat shock response (HSR) resulting in cellular survival. Present review concentrated on the cellular survival against heat stress employing the harmonized impact of σE and σH regulons and the HSPs as well as their inter connectivity towards the maintenance of cellular survival.

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Structural determinants stabilizing the axial channel of ClpP for substrate translocation

Abstract: Summary Acyldepsipeptides (ADEPs) antibiotics bind to

Cited by 24 publications

References 43 publications

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

Mechanism to control the cell lysis and the cell survival strategy in stationary phase under heat stress

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