Helix Unfolding/Refolding Characterizes the Functional Dynamics of Staphylococcus aureus Clp Protease

Ye, Fei; Zhang, Jie; Liu, Hongchuan; Hilgenfeld, Rolf; Zhang, Ruihan; Kong, Xiangqian; Li, Lianchun; Lu, Junyan; Zhang, Xinlei; Li, Donghai; Jiang, Hualiang; Yang, Cai‐Guang; Luo, Cheng

doi:10.1074/jbc.m113.452714

Cited by 49 publications

(64 citation statements)

References 53 publications

(71 reference statements)

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“…6a). Protein NMR studies with EcClpP 35 have revealed the presence of at least two states in solution, which is supported by molecular dynamics simulations 36 and normal mode analysis 37 suggesting that ClpP samples different conformations. We sought to elucidate the structural basis of ADEP allostery by studying a panel of mutant proteins, which adopt different conformational states.…”

Section: Saclpp+u1 (%)mentioning

confidence: 83%

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

et al. 2015

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The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA þ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

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Section: Saclpp+u1 (%)mentioning

confidence: 83%

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

et al. 2015

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“…In addition, the axial loops interact with the proximal surface of the bound ClpX heptamer (and presumably ClpA/C) to prevent motions that might interfere with polypeptide translocation (48). Conformational dynamics at the tetradecamer interface have also been revealed by crystallization of S. aureus and other ClpPs in two different states, one with all seven of the "handle domains" (a long ␣␤-extension consisting of strand-9 and helix-E) extended and making multiple contacts with the apposing heptamers (closed state) and one with the handle domains contracted and folded away from the apposing heptamer leaving gaps at the interface large enough for peptides to pass (open state) (46,49). Heptamers in the contracted state are not active because the orientation of the catalytic residues depends on a hydrogen-bonding network affected by interactions with the handles from an adjacent subunit and from an apposing subunit across the tetradecamer (44).…”

Section: Discussionmentioning

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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“…Derivate mit nur einem Halogen-Substituenten an verschiedenen Positionen (3,5,8,9)z eigten ein reduziertes Aktivierungs-Potenzial. Das erste Set (1-13)u mfasst Va riationen des Benzylsubstitutions-Musters von D9.…”

Section: Angewandte Chemieunclassified

“…Eine Kokristallisation der Wildtyp-hClpP mit D9 war zunächst nicht mçglich. [8,27] Die Hçhe des hClpP-Tetradekamers ist um etwa 10 zum Wildtyp verringert, was durch eine verkürzte E-Helix an der Schnittstelle der beiden heptameren Ringe erklärt werden kann (Abbildung 4a,c). [11] Wirk lonierten und reinigten die entsprechende humane Mutante hClpP Y118A, die eine verstärkte Peptidase-Aktivitätauch ohne D9 zeigte.…”

Section: Angewandte Chemieunclassified

“…[3] hClpP nimmt die Form zweier heptamerer Ringe an, die hydrophobe Interaktionsflächen rund um deren apikale Poren aufweisen und so die Bindung an die LGF-Schleifen des hClpX-Chaperons ermçglichen. [7][8][9] Ein Hauptmerkmal dieser Ausprägungen ist die Orientierung der zentralen E-Helix, die kritische Kontakte zwischen den Heptamer-Ringen in der tetradekameren, gestreckten Va riante bildet und direkt mit dem aktiven Zentrum verbunden ist. [5] Die hClpX-Bindung stabilisiert einen tetradekameren Zustand von ClpP,d ie ansonsten inaktive Heptamere in vitro bildet.…”

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Selektive Aktivierung der humanen caseinolytischen Protease P (ClpP)

et al. 2018

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Die caseinolytische Protease P( ClpP) ist die proteolytische Komponente des Protein-Abbau-Komplexes ClpXP.Ihre genaue Funktion und Regulation sind grçßtenteils unerforscht. Hier stellen wir eine niedermolekulare Verbindung (D9)v or,d ie durchN achahmung des natürlichen Chaperons ClpX als potenter und Spezies-selektiver Aktivator der humanen ClpP (hClpP) wirkt. Struktur-Aktivitäts-Studien hoben die Wichtigkeit eines halogenierten Benzyl-Motivs innerhalb von D9 hervor.D ieses interagiert mit einem einzigartigen aromatischen Aminosäure-Netzwerk in hClpP.M utations-und Strukturstudien legen nahe,d ass dieses sogenannte YYW-Motiv die hClpP-Aktivitäts treng kontrolliert und den Substratumsatz durchI nteraktion mit passenden Liganden reguliert. Ferner ist dieses Motiv besonders fürC lpP hçherer Organismen charakteristischu nd existiert nichti ng etesteten bakteriellen Homologen. Dies erlaubt eine Spezies-selektive Analyse,womit D9 ein vielseitiges Werkzeug fürdie Untersuchung von mechanistischen Eigenschaften der hClpP werden kann.

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Helix Unfolding/Refolding Characterizes the Functional Dynamics of Staphylococcus aureus Clp Protease

Cited by 49 publications

References 53 publications

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

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

Selektive Aktivierung der humanen caseinolytischen Protease P (ClpP)

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