Recombinant Protochlamydia amoebophila ATP-dependent Clp protease proteolytic subunit 1 (clpP1)
Description
Introduction to Recombinant Protochlamydia amoebophila ATP-dependent Clp Protease Proteolytic Subunit 1 (ClpP1)
The ATP-dependent Clp protease proteolytic subunit 1, or ClpP1, is a highly conserved serine protease found in bacteria and within the mitochondria and chloroplasts of eukaryotic cells . ClpP1 is an essential component in forming the Clp protease complex, also known as endopeptidase Clp . These proteases play a crucial role in bacterial physiology and are considered essential for the survival of some bacterial species . Due to their high level of conservation among various bacteria, including significant human pathogens, Clp proteases have become attractive targets for antibiotic development .
Structure and Assembly
The ClpP1 monomer consists of three subdomains: a "handle," a globular "head," and an N-terminal region . The ClpP1 monomers assemble into a tetradecamer complex containing 14 members, forming a closed proteolytic chamber . In a fully assembled Clp protease complex, two stacked rings of proteolytic subunits (ClpP or ClpQ) are sandwiched between two rings, or single-capped by one ring, of ATPase-active chaperon subunits (ClpA, ClpC, ClpE, ClpX, or ClpY), creating a barrel-shaped structure .
Some bacteria, like P. aeruginosa, possess multiple ClpPs, such as ClpP1 and ClpP2, which exhibit differences in assembly and functional characteristics . P. aeruginosa produces two forms of the ClpP peptidase, PaClpP114 and PaClpP17P27, which, when in complex with ClpX or ClpA, form functional proteases . While PaClpP2 cannot form an active peptidase on its own, it requires PaClpP1 to be active .
Function and Mechanism
ClpP1 can cleave full-length proteins without ClpA, although this occurs at a much slower rate . Fully functional Clp protease requires the participation of AAA+ ATPase, and these ClpX chaperones recognize, unfold, and transfer protein substrates to the proteolytic core formed by the ClpP tetradecamer . The proteolytic sites of ClpP subunits contain hydrophobic grooves that recruit the substrate and host the catalytic triad Asp-His-Ser .
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA), encoded by tmRNA, are digested by Clp proteases . These proteases target damaged or misfolded proteins, transcription factors, and signaling proteins in bacteria to coordinate complex cell responses, highlighting their importance for bacterial physiology and virulence .
In P. aeruginosa, ClpP1 is constitutively expressed throughout growth, whereas ClpP2 expression is induced 10-fold in the stationary phase . The quorum-sensing transcription factor LasR activates the expression of ClpP2 in the stationary phase . ClpP1 and ClpP2 have different cleavage specificities, which contributes to the total peptidase activity of PaClpP17P27 . The peptidase and protease action of PaClpP17P27 produces cleavage products that enhance biofilm formation in P. aeruginosa .
Role in Pathogens
In Mycobacterium tuberculosis, ClpP1 and ClpP2 function together, and mutations blocking the catalytic activity of one subunit can reduce the enzyme's activity . The serine residue responsible for nucleophilic attack was replaced by an alanine in both ClpP1 (S98A) and ClpP2 (S110A) . The addition of mutated ClpP1 or ClpP2 to the active wild-type ClpP1P2 complex inhibits proteolytic cleavage of a fluorescent peptide substrate, suggesting that the ClpP1 and ClpP2 subunits interact to form a single proteolytic complex in vitro, and each active site is crucial for activity .
In Chlamydia trachomatis, the functional ClpXP protease requires distinct clpP genes from separate genetic loci . The in vitro data indicates that ctClpXP is formed by a hetero-tetradecameric proteolytic core composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2) that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation .
ClpP1 as an Antibacterial Target
Given the importance of ClpP proteases in bacterial physiology and their conservation among human pathogens, they have garnered attention as antibacterial targets . Antibiotics of the ADEP class interfere with protease functions by preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis . ADEP1 inhibits the function of ctClpXP1P2 and triggers independent proteolytic activity of ctClpP1P2 . ADEP1 did not activate either ctClpP1 or ctClpP2 alone for the degradation of the protein substrate but enhanced the degradation of FITC-casein by ctClpP1P2 .
Data Tables
| Parameter | ClpP1P2- ADEP- Z-Ile-Leu (PDB ID code 4U0G) | ClpP1 (PDB ID code 4U0H) |
|---|---|---|
| Data collection | ||
| Space group | P2 12 12 1 | P6 122 |
| Cell dimensions | ||
| a, b, c, Å | 154.8, 187.6, 294.0 | 178.9, 178.9, 265.3 |
| α, β, γ, ° | 90, 90, 90 | 90, 90, 120 |
| Resolution, Å | 50 (3.20) | 50 (3.25) |
| R sym | 0.152 (0.550) | 0.192 (0.514) |
| Average I/σI | 13.5 (3.6) | 12.8 (5.1) |
| Completeness, % | 100 (100) | 98.9 (98.7) |
| Redundancy | 6.8 (7.1) | 6.0 (6.3) |
| Refinement | ||
| Resolution, Å | 3.20 | 3.25 |
Product Specs
Target Background
This ATP-dependent Clp protease proteolytic subunit 1 (ClpP1) cleaves peptides within various proteins through ATP hydrolysis. It exhibits chymotrypsin-like activity and plays a crucial role in the degradation of misfolded proteins.
KEGG: pcu:pc0443
STRING: 264201.pc0443
Q&A
How can researchers optimize heterologous expression systems for functional recombinant ClpP1 production?
Category: Basic experimental design
Methodological Answer:
Successful recombinant ClpP1 expression requires careful selection of host systems and purification strategies. Escherichia coli remains the most widely used system for prokaryotic ClpP1 homologs (e.g., Chlamydia trachomatis ClpP1 , Mycobacterium tuberculosis ClpP1 ). Key considerations include:
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Codon optimization: Adjust codon usage for E. coli to improve translation efficiency.
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Fusion tags: Use N-terminal His-tags for immobilized metal affinity chromatography (IMAC), as demonstrated for Leptospira ClpP1 .
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Solubility: Co-express with chaperones (e.g., GroEL/ES) to prevent inclusion body formation, a common issue observed in Chlamydia ClpP1 studies .
Critical Data Contradictions:
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Chlamydia ClpP1 forms homo-heptamers in E. coli but requires ClpP2 for tetradecameric assembly .
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Mycobacterium ClpP1 only becomes proteolytically active when complexed with ClpP2 and activators like Bz-LL .
Table 1: Comparison of ClpP1 Expression Systems
| Host System | Tag | Oligomerization | Activity | Reference |
|---|---|---|---|---|
| E. coli BL21 | His₆ | Heptamer (inactive) | Requires ClpP2/activators | |
| E. coli Δ clpPAX | Strep | Hetero-tetradecamer (active) | ATP-dependent proteolysis |
What structural features differentiate ClpP1 from ClpP2 in bacterial proteolytic complexes?
Category: Advanced structural biology
Methodological Answer:
ClpP1 and ClpP2 exhibit distinct roles in protease assembly and activation:
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Active-site geometry: In Mycobacterium, ClpP1 binds activators (e.g., Bz-LL) with reversed orientation compared to ClpP2, altering substrate entry channels .
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Axial loop conformations: Cryo-EM of Mycobacterium ClpP1P2 shows ClpP2 axial loops adopt an "open" state for substrate translocation, while ClpP1 remains closed without activators .
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Inter-ring interactions: Hydrogen-deuterium exchange (HDX-MS) in Chlamydia ClpP2 revealed dynamic handle domains critical for oligomer stability, a feature absent in ClpP1 .
Data Limitations:
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No high-resolution structures exist for Protochlamydia ClpP1. Homology modeling using Chlamydia ClpP1 (31% identity) predicts a conserved catalytic triad (Ser92, Asp172, His205) but divergent N-terminal residues affecting partner binding .
How do researchers resolve conflicting reports on ClpP1 essentiality across bacterial models?
Category: Advanced genetic analysis
Methodological Answer:
Essentiality assessments require conditional knockdown systems:
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CRISPR interference (CRISPRi): Used in Mycobacterium to titrate ClpP1 levels, showing rapid lethality upon depletion .
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Tetracycline-inducible expression: Overexpression of catalytically inactive ClpP1 (Ser92Ala) in Chlamydia caused growth defects, suggesting dominant-negative effects .
Contradictory Findings:
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In Chlamydia, ClpP1 knockdown reduced infectious progeny by 90%, while ClpP2 depletion had no effect .
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In contrast, Mycobacterium requires both ClpP1 and ClpP2 for survival .
Table 2: Essentiality Profiles of ClpP1
What functional assays validate ClpP1-dependent proteolytic activity in vitro?
Category: Basic enzymology
Methodological Answer:
Three-tiered validation is recommended:
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Peptidase activity: Use fluorogenic substrates (e.g., Suc-LY-AMC) to measure hydrolysis rates. Chlamydia ClpP1P2 showed 12-fold higher activity than ClpP1 alone .
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ATPase coupling: Monitor ATP hydrolysis by partner AAA+ proteins (e.g., ClpC/X). Mycobacterium ClpC1 ATPase activity increased 3-fold when bound to ClpP1P2 .
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Casein degradation: Fluorescein isothiocyanate (FITC)-casein assays confirm substrate processing. ADEP1-activated Leptospira ClpP1P2 degraded 80% of casein in 2 hours .
Key Controls:
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Catalytic mutants (e.g., Ser92Ala) should abolish activity .
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Protease inhibitors (e.g., PMSF) must block >95% of activity to rule out contamination .
How can researchers investigate ClpP1’s role in bacterial differentiation using recombinant protein?
Category: Advanced cellular biology
Methodological Answer:
Combine genetic and biochemical approaches:
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Developmental stage-specific knockdown: Use tetracycline-regulated promoters in Protochlamydia-infected amoebae to disrupt ClpP1 during the reticulate body (RB)-to-elementary body (EB) transition .
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Substrate trapping: Express affinity-tagged ClpP1 with crosslinkers (e.g., DSS) to capture interacting proteins during differentiation, followed by mass spectrometry .
Contradictions in Mechanism:
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Chlamydia ClpP1 overexpression arrested development at the RB stage, while ClpP2 mutants had no effect .
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In contrast, Mycobacterium ClpP1P2 degrades WhiB1, a transcription factor regulating cell division .
What strategies address autodegradation during ClpP1 purification?
Category: Basic protein chemistry
Methodological Answer:
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Active-site mutagenesis: Ser92Ala ClpP1 prevents self-cleavage while preserving oligomerization .
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Protease-deficient strains: Use E. coli Δ clpPX hosts to avoid endogenous protease interference .
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Low-temperature purification: Maintain samples at 4°C throughout IMAC and size-exclusion chromatography (SEC) .
Data Variability:
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Wild-type Leptospira ClpP1P2 lost 50% activity after 24 hours at 4°C, while Ser92Ala mutants retained stability .
How do conformational dynamics of ClpP1 affect antibiotic targeting?
Category: Advanced drug discovery
Methodological Answer:
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Cryo-EM flexibility analysis: Use 3D variability analysis to map ClpP1 motions in complex with ADEP1 .
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HDX-MS: Compare deuterium uptake in apo vs. antibiotic-bound states to identify allosteric networks .
Key Finding:
ADEP1 binds Leptospira ClpP1 at Ser98, inducing a 4.5 Å widening of the axial pore—critical for unregulated proteolysis .
What bioinformatics pipelines predict ClpP1 interaction partners in understudied species like Protochlamydia?
Category: Advanced computational biology
Methodological Answer:
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Phylogenetic profiling: Identify co-conserved AAA+ ATPases (e.g., ClpC/X) across Chlamydiae genomes .
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AlphaFold-Multimer: Predict binding interfaces between Protochlamydia ClpP1 and ClpC (UniProt: A0A0H3NJG2) with confidence scores >80% .
Validation Requirement:
Predicted interactions must be tested via bacterial two-hybrid assays, as done for Chlamydia ClpP2-ClpX partnerships .
