Structure of the central Staphylococcus aureus AAA+ protease MecA/ClpC/ClpP

Bacterial AAA+ proteases are composed of a AAA+ partner (e.g. ClpC) and an associated peptidase (e.g. ClpP). They represent ATP-fuelled and self-compartmentalized proteolytic machines that are crucial for stress resistance and virulence. ClpC requires cooperation with adaptor proteins such as MecA for activation and complex formation with ClpP. Here, we present the cryo-EM structure of the MecA/ClpC/ClpP complex from the major pathogen Staphylococcus aureus. MecA forms a dynamic crown on top of the ClpC/ClpP complex with its substrate-binding domain positioned near the ClpC pore site, likely facilitating substrate transfer. ClpC/ClpP complex formation involves ClpC P-loops and ClpP N-terminal β-hairpins, which insert into the central ClpC threading channel and contact sites next to the ClpC ATPase center. ClpC and ClpP interactions are asymmetric and dictated by the activity states of ClpC ATPase subunits. ClpP binding increases ClpC ATPase and threading activities in a β-hairpin dependent manner, illuminating an allosteric pathway in the cooperation of ATPase and peptidase components in bacterial AAA+ proteases.

• Publication year

  2025

• Venue

  bioRxiv

• Publication date

  2025-06-06

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1038/s42003-025-08908-wPMID 41087538PMCID 12521514
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Structural Insights into Bortezomib-Induced Activation of the Caseinolytic Chaperone-Protease System in Mycobacterium tuberculosis

  2025influential reference
• Activation Mechanism and Structural Assembly of the Mycobacterium tuberculosis ClpP1P2 Protease and Its Associated ATPases

  2025cited by this paper
• Structural and mechanistic studies on human LONP1 redefine the hand-over-hand translocation mechanism

  2024cited by this paper
• MdfA is a novel ClpC adaptor protein that functions in the developing Bacillus subtilis spore

  2024cited by this paper
• Structural insights into the Clp protein degradation machinery

  2024influential reference
• Accurate structure prediction of biomolecular interactions with AlphaFold 3

  2024influential reference
• 3DFlex: determining structure and motion of flexible proteins from cryo-EM

  2023cited by this paper
• Improvement of cryo-EM maps by simultaneous local and non-local deep learning

  2023cited by this paper
• AlphaFill: enriching AlphaFold models with ligands and cofactors

  2022cited by this paper
• Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules

  2022cited by this paper
• Benchmarking the Accuracy of AlphaFold 2 in Loop Structure Prediction

  2022cited by this paper
• Structure of the drug target ClpC1 unfoldase in action provides insights on antibiotic mechanism of action

  2022cited by this paper
• AAA+ protease-adaptor structures reveal altered conformations and ring specialization

  2022cited by this paper
• Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation

  2021cited by this paper
• BacPROTACs mediate targeted protein degradation in bacteria

  2021cited by this paper
• Highly accurate protein structure prediction with AlphaFold

  2021cited by this paper
• 3D Variability Analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM

  2020cited by this paper
• Stairway to translocation: AAA+ motor structures reveal the mechanisms of ATP‐dependent substrate translocation

  2020cited by this paper
• Processive extrusion of polypeptide loops by a Hsp100 disaggregase

  2020cited by this paper
• Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

  2020cited by this paper
• Structural basis for distinct operational modes and protease activation in AAA+ protease Lon

  2020cited by this paper
• DeepEMhancer: a deep learning solution for cryo-EM volume post-processing

  2020cited by this paper
• ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

  2020cited by this paper
• Neural network particle picking and denoising in cryoEM with Topaz

  2020cited by this paper
• The molecular principles governing the activity and functional diversity of AAA+ proteins

  2019cited by this paper
• Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

  2019cited by this paper
• Roles of the ClpX IGF loops in ClpP association, dissociation, and protein degradation

  2019cited by this paper
• Cryo-EM structure of the ClpXP protein degradation machinery

  2019influential reference
• Toxic Activation of an AAA+ Protease by the Antibacterial Drug Cyclomarin A.

  2019cited by this paper
• Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

  2019cited by this paper
• Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

  2019cited by this paper
• Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

  2019cited by this paper
• A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery

  2019cited by this paper
• Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing.

  2019influential reference
• Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis

  2019influential reference
• ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps

  2018cited by this paper
• Reversible inhibition of the ClpP protease via an N-terminal conformational switch

  2018influential reference
• Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

  2018cited by this paper
• Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase

  2018cited by this paper
• Regulated Proteolysis in Bacteria.

  2018cited by this paper
• Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation

  2018cited by this paper
• Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome

  2018cited by this paper
• Structure and Function of the 26S Proteasome.

  2018cited by this paper
• Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase

  2017cited by this paper
• Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing

  2017cited by this paper
• Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

  2017cited by this paper
• AAA-ATPases in Protein Degradation

  2017cited by this paper
• Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104

  2017cited by this paper
• Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis*

  2016cited by this paper
• Gates, Channels, and Switches: Elements of the Proteasome Machine.

  2016cited by this paper
• AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

  2015cited by this paper
• Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

  2015cited by this paper
• Dynamics of the ClpP serine protease: A model for self-compartmentalized proteases

  2014cited by this paper
• Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

  2014cited by this paper
• E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

  2013cited by this paper
• Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus.

  2013cited by this paper
• Insights into Structural Network Responsible for Oligomerization and Activity of Bacterial Virulence Regulator Caseinolytic Protease P (ClpP) Protein*

  2012cited by this paper
• Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine

  2012cited by this paper
• Rational design of true monomeric and bright photoactivatable fluorescent proteins

  2012influential reference
• AAA+ proteases: ATP-fueled machines of protein destruction.

  2011cited by this paper
• Structural Switching of Staphylococcus aureus Clp Protease

  2011cited by this paper
• The natural product cyclomarin kills Mycobacterium tuberculosis by targeting the ClpC1 subunit of the caseinolytic protease.

  2011cited by this paper
• Structure and mechanism of the hexameric MecA–ClpC molecular machine

  2011cited by this paper
• ClpX(P) generates mechanical force to unfold and translocate its protein substrates.

  2011cited by this paper
• Features and development of Coot

  2010cited by this paper
• Structures of ClpP in complex with acyldepsipeptide antibiotics reveal its activation mechanism

  2010cited by this paper
• Binding of the ClpA Unfoldase Opens the Axial Gate of ClpP Peptidase*

  2010cited by this paper
• Acyldepsipeptide antibiotics induce the formation of a structured axial channel in ClpP: A model for the ClpX/ClpA-bound state of ClpP.

  2010cited by this paper
• Molecular Determinants of MecA as a Degradation Tag for the ClpCP Protease*

  2009influential reference
• Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

  2009cited by this paper
• Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases.

  2008cited by this paper
• Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry.

  2007cited by this paper
• Distinct static and dynamic interactions control ATPase-peptidase communication in a AAA+ protease.

  2007cited by this paper
• The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes.

  2006cited by this paper
• Dysregulation of bacterial proteolytic machinery by a new class of antibiotics

  2005cited by this paper
• Characterization of the photoconversion on reaction of the fluorescent protein Kaede on the single-molecule level.

  2005cited by this paper
• Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP.

  2004cited by this paper
• Communication between ClpX and ClpP during substrate processing and degradation

  2004influential reference
• MecA, an adaptor protein necessary for ClpC chaperone activity

  2003cited by this paper
• Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase

  2001cited by this paper
• Functional Domains of the ClpA and ClpX Molecular Chaperones Identified by Limited Proteolysis and Deletion Analysis*

  2001cited by this paper
• The N‐ and C‐terminal domains of MecA recognize different partners in the competence molecular switch

  1999cited by this paper

• No citing papers are available for this paper.