Recombinant Protochlamydia amoebophila Leucine--tRNA ligase (leuS), partial

What is Protochlamydia amoebophila and why is its Leucyl-tRNA synthetase significant?

Protochlamydia amoebophila is an obligate intracellular bacterium belonging to the Chlamydiae group that exists as an endosymbiont of amoebae. Its Leucyl-tRNA synthetase (LeuRS/leuS) is significant because it represents a crucial component of the protein synthesis machinery in this organism. LeuRS catalyzes the ATP-dependent ligation of L-leucine to tRNA(Leu), an essential step in translation .

The study of P. amoebophila LeuRS provides valuable insights into:

• The evolution of aminoacyl-tRNA synthetases in intracellular bacteria

• Adaptations specific to obligate intracellular lifestyles

• Potential targets for antimicrobial development

• Mechanisms of host-symbiont coevolution at the molecular level

How does Protochlamydia amoebophila LeuRS compare structurally to other bacterial LeuRS enzymes?

Based on primary sequence analysis, LeuRS enzymes can be divided into bacterial and archaeal/eukaryotic types. Like other bacterial LeuRSs, P. amoebophila LeuRS contains several key domains :

• Rossmann-fold domain (for amino acid activation and tRNA charging)

• CP1 domain (for editing)

• α-helix bundle domain (for tRNA binding)

• C-terminal domain (CTD, for tRNA binding)

What are the recommended protocols for cloning and expressing recombinant P. amoebophila LeuRS?

For successful cloning and expression of recombinant P. amoebophila LeuRS, researchers should follow these methodological steps:

• Gene Amplification:

  • Design primers based on the sequenced genome of P. amoebophila

  • Use high-fidelity polymerase for PCR amplification

  • Consider codon optimization if expressing in E. coli

• Vector Selection and Cloning:

  • For bacterial expression, pET-series vectors (e.g., pET28a) provide a robust expression system with N-terminal His-tags

  • Digest with appropriate restriction enzymes (commonly NdeI/BamHI or NheI/BamHI)

  • Ligate the PCR product into the pre-cleaved vector

• Expression Conditions:

  • Transform into an E. coli expression strain (BL21(DE3) or derivatives)

  • Culture at lower temperatures (16-25°C) after induction to enhance solubility

  • Use auto-induction media or IPTG induction (0.1-0.5 mM)

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing and buffer exchange

Similar protocols have been successfully used for related LeuRS enzymes from other organisms , though adaptations may be necessary for optimal expression of P. amoebophila LeuRS.

What assays can be used to measure the activity of recombinant P. amoebophila LeuRS?

Several complementary assays can be employed to characterize the aminoacylation and editing activities of recombinant P. amoebophila LeuRS:

• Aminoacylation Activity Assays:

  • ATP-PPi Exchange Assay: Measures the first step of the reaction (amino acid activation)

  • [32P]-labeled tRNA Aminoacylation Assay: Tracks the formation of aminoacyl-tRNA over time

  • Pyrophosphate Release Assay: A coupled continuous assay monitoring pyrophosphate release

• Editing Activity Assays:

  • Deacylation Assay: Measures the hydrolysis of pre-formed mischarged tRNAs

  • AMP Formation Assay: Detects the release of AMP during pre-transfer editing

  • Thin-Layer Chromatography (TLC): Separates aminoacyl-tRNA from free amino acids

• tRNA Binding Assays:

  • Electrophoretic Mobility Shift Assay (EMSA): Assesses protein-tRNA complex formation

  • Surface Plasmon Resonance (SPR): Determines binding kinetics and affinity

These methods have been applied to study aminoacyl-tRNA synthetases from various organisms and can be adapted for P. amoebophila LeuRS .

How is P. amoebophila LeuRS phylogenetically related to other LeuRS enzymes?

Phylogenetic analysis of LeuRS sequences reveals important evolutionary relationships:

• Major LeuRS Types:

  • LeuRS can be divided into bacterial type (found in bacteria and organelles) and archaeal/eukaryotic type (found in eukaryotes and most archaea)

  • P. amoebophila LeuRS belongs to the bacterial type, consistent with its bacterial origin

• Position in Chlamydiae:

  • Among Chlamydiae, Protochlamydia represents an early-branching lineage

  • Analysis of LeuRS sequences supports the monophyly of the Chlamydiae group

  • P. amoebophila LeuRS shares sequence signatures specific to the Chlamydiae clade

• Horizontal Gene Transfer Considerations:

  • Chlamydiae have contributed at least 55 genes to Plantae genomes through horizontal gene transfer

  • No evidence suggests that the LeuRS gene specifically was transferred, but this demonstrates the prevalence of gene transfer events involving Chlamydiae

• Coevolution with tRNA:

  • The P. amoebophila LeuRS has coevolved with its cognate tRNA^Leu molecules

  • This coevolution has shaped the specificity determinants in both the enzyme and tRNA

What evidence exists for horizontal gene transfer involving P. amoebophila genes?

Extensive phylogenomic analyses have identified significant horizontal gene transfer (HGT) events involving Chlamydiae genes:

• Plant Genomes:

  • At least 55 Chlamydiae-derived genes have been identified in algae and plants

  • 67% (37/55) of these genes encode proteins with putative plastid targeting signals

  • 3 genes encode proteins with mitochondrial functions

• Transfer Mechanisms:

  • Evidence supports both endosymbiotic gene transfer and a "horizontal gene transfer ratchet" driven by recurrent endoparasitism

  • The distribution of Chlamydiae genes across Plantae species suggests ancient transfer events

• Functional Significance:

  • Transferred genes extend beyond plastid metabolism, suggesting broader contributions to eukaryotic cell functions

  • Some transferred genes have undergone functional adaptation in their new hosts

While LeuRS specifically has not been identified as a transferred gene, the extensive history of gene transfer from Chlamydiae provides important context for understanding the evolutionary history of P. amoebophila genes.

What are the key substrate recognition and catalytic residues in P. amoebophila LeuRS?

Based on comparative analysis with other LeuRS enzymes, several key residues and motifs are likely critical for P. amoebophila LeuRS function:

• Amino Acid Recognition:

  • A leucine-specific binding pocket within the Rossmann fold domain

  • Conserved motifs HIGH and KMSKS typical of class I aminoacyl-tRNA synthetases

  • Residues that interact with the side chain of leucine to discriminate it from other amino acids

• tRNA Recognition Elements:

  • The C-terminal domain (CTD) contains critical residues for tRNA binding

  • In archaeal LeuRS, residues like Asp845, Ile849, Pro962, Ile964, Ile966, and Glu967 in the CTD are essential for tRNA recognition

  • The P. amoebophila enzyme likely has analogous residues that recognize specific features of its cognate tRNA^Leu

• Editing Domain:

  • The CP1 domain contains residues crucial for hydrolytic editing

  • This domain prevents misacylation by removing incorrectly attached amino acids

  • Conserved threonine residues that form hydrogen bonds with the mischarged amino acid

• Catalytic Core:

  • ATP binding residues in the Rossmann fold

  • Metal ion coordination sites (typically Mg²⁺) that facilitate catalysis

  • Residues that position the 3' end of the tRNA for aminoacylation

The specific roles of these residues would need to be confirmed through mutagenesis and structural studies of P. amoebophila LeuRS.

How does the metabolic state of P. amoebophila affect LeuRS activity?

The metabolic state of P. amoebophila significantly impacts protein synthesis and likely affects LeuRS activity:

• Elementary Body (EB) Stage:

  • Contrary to previous assumptions, P. amoebophila EBs maintain metabolic activity, including D-glucose metabolism and respiratory activity

  • Protein synthesis occurs in EBs, requiring functional LeuRS and other aminoacyl-tRNA synthetases

  • Metabolic activity in EBs is critical for maintaining infectivity

• Energy Requirements:

  • LeuRS requires ATP for amino acid activation

  • The metabolic activity observed in P. amoebophila EBs suggests that ATP generation continues in this stage

  • The pentose phosphate pathway and TCA cycle activity in EBs provide energy for processes including protein synthesis

• Regulation during Life Cycle:

  • LeuRS activity may be differentially regulated during the developmental cycle

  • The transition between replicative and elementary body forms likely involves changes in translation rates and potentially LeuRS expression or activity

• Nutrient Availability Impact:

  • D-glucose availability is essential for sustaining metabolic activity in P. amoebophila EBs

  • Nutrient deprivation leads to rapid decline in infectivity, suggesting an impact on protein synthesis machinery including LeuRS

This relationship between metabolism and LeuRS activity has implications for understanding P. amoebophila's biology and potential therapeutic targets.

What potential role does P. amoebophila LeuRS play in human disease pathogenesis?

P. amoebophila and its essential proteins like LeuRS may have implications for human health:

• Association with Pneumonia:

  • P. amoebophila has been detected in bronchoalveolar lavage samples from patients with pneumonia

  • The organism is likely resistant to human alveolar macrophages, suggesting potential for causing infection

  • Specific PCR targeting P. amoebophila has been developed for diagnostic purposes

• LeuRS as a Drug Target:

  • As an essential enzyme for protein synthesis, LeuRS represents a potential therapeutic target

  • Inhibitors targeting bacterial LeuRS could potentially treat P. amoebophila infections

  • The structural differences between bacterial and human LeuRS could be exploited for selective inhibition

• Antibiotic Resistance Considerations:

  • Mutations in LeuRS could potentially confer resistance to antibiotics that target this enzyme

  • Understanding the structure and function of P. amoebophila LeuRS could help predict resistance mechanisms

• Diagnostic Applications:

  • The gene encoding LeuRS could serve as a target for molecular diagnostics

  • Species-specific regions of the LeuRS gene might enable sensitive and specific detection of P. amoebophila

How can structural studies of P. amoebophila LeuRS contribute to drug development?

Structural characterization of P. amoebophila LeuRS offers several avenues for therapeutic development:

• Structure-Based Drug Design:

  • Crystal structures or homology models of P. amoebophila LeuRS can guide rational drug design

  • Key binding pockets can be targeted for small molecule inhibitors

  • Virtual screening against structural models can identify lead compounds

• Exploitable Structural Differences:

  • Comparative analysis between bacterial and human LeuRS structures can identify unique features

  • The aminoacylation active site, editing domain, and tRNA binding interface all represent potential targets

  • Bacterial-specific insertions or deletions may provide selectivity

• Allosteric Inhibition Strategies:

  • Identification of allosteric sites unique to bacterial LeuRS

  • Development of compounds that disrupt enzyme dynamics rather than blocking the active site

  • Potential for overcoming resistance mechanisms that affect active site inhibitors

• Fragment-Based Approaches:

  • Use of fragment libraries to identify small molecules that bind to different regions of LeuRS

  • Fragment growing or linking strategies to develop high-affinity inhibitors

  • NMR or X-ray crystallography to validate fragment binding

What are the main challenges in expressing and purifying recombinant P. amoebophila LeuRS?

Researchers face several technical challenges when working with recombinant P. amoebophila LeuRS:

• Solubility Issues:

  • Large multi-domain proteins like LeuRS often have solubility problems

  • Solution: Use solubility-enhancing fusion tags (SUMO, MBP, TrxA), lower induction temperatures (16-20°C), and specialized E. coli strains (Rosetta, Arctic Express)

• Protein Stability:

  • LeuRS may exhibit limited stability in vitro after purification

  • Solution: Optimize buffer conditions (add glycerol, reduce salt concentration), include stabilizing ligands (ATP, leucine), and store with protease inhibitors

• Post-Translational Modifications:

  • Bacterial expression systems may not reproduce native modifications

  • Solution: Consider eukaryotic expression systems if modifications are critical, or use cell-free systems for expression

• Functional Activity:

  • Recombinant enzyme may have reduced activity compared to native protein

  • Solution: Ensure proper folding through controlled refolding protocols if necessary, and verify activity using sensitive assays

• tRNA Substrate Availability:

  • Testing LeuRS activity requires cognate tRNA^Leu

  • Solution: In vitro transcription of tRNA genes using T7 RNA polymerase, as demonstrated in related studies

What methods are most effective for analyzing the interaction between P. amoebophila LeuRS and its cognate tRNA?

Several complementary techniques can effectively characterize LeuRS-tRNA interactions:

• Biochemical Approaches:

  • Gel Mobility Shift Assays: Detect complex formation between LeuRS and tRNA

  • Filter Binding Assays: Quantify binding affinity (Kd values)

  • Footprinting Analysis: Identify protected regions of tRNA when bound to LeuRS

  • Crosslinking Studies: Map interaction sites between protein and RNA

• Biophysical Methods:

  • Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of binding

  • Surface Plasmon Resonance (SPR): Measure association/dissociation kinetics

  • Microscale Thermophoresis (MST): Analyze interactions in solution with minimal sample consumption

  • Analytical Ultracentrifugation: Characterize complex formation and stoichiometry

• Structural Approaches:

  • X-ray Crystallography: Determine high-resolution structures of the complex

  • Cryo-Electron Microscopy: Visualize the complex without crystallization

  • NMR Spectroscopy: Map interaction interfaces in solution

  • Small-Angle X-ray Scattering (SAXS): Obtain low-resolution structural information

• Computational Methods:

  • Molecular Docking: Predict binding modes in silico

  • Molecular Dynamics Simulations: Analyze dynamic aspects of the interaction

  • Sequence Covariation Analysis: Identify co-evolving residues between LeuRS and tRNA

The combination of these approaches provides comprehensive insights into the specific recognition mechanism between P. amoebophila LeuRS and its cognate tRNA.

How does P. amoebophila LeuRS compare to LeuRS systems in other intracellular bacteria?

Comparative analysis reveals important similarities and differences between P. amoebophila LeuRS and other bacterial systems:

This comparison highlights both the conservation of core LeuRS function across bacterial species and the potential adaptations specific to obligate intracellular lifestyle in P. amoebophila.

What can be learned about P. amoebophila LeuRS from unique bacterial LeuRS systems like those in Natranaerobius?

The unique features of LeuRS systems from other bacteria provide valuable insights for understanding P. amoebophila LeuRS:

• Lessons from Natranaerobius System:

  • Natranaerobius species contain two distinct LeuRS types (LeuRS1 and LeuRS2)

  • These systems demonstrate how bacterial-type LeuRS can recognize archaeal-type tRNA^Leu

  • This has relevance for P. amoebophila, which may have adapted its LeuRS to recognize specific tRNA features during evolution

• Domain Architecture Insights:

  • Studies of Natranaerobius LeuRS revealed the importance of the C-terminal domain (CTD) in tRNA recognition

  • The role of specific domains can be extrapolated to predict critical regions in P. amoebophila LeuRS

• Editing Function Comparison:

  • Natranaerobius LeuRS demonstrated that editing function is maintained even when the CTD is deleted

  • This suggests functional independence of domains that may be relevant to P. amoebophila LeuRS

• Evolutionary Implications:

  • Coexistence of bacterial LeuRS with archaeal tRNAs in Natranaerobius provides a model for understanding enzyme-substrate coevolution

  • This may inform hypotheses about the adaptation of P. amoebophila LeuRS to its specific cellular environment

What are the most promising areas for future research on P. amoebophila LeuRS?

Several high-priority research directions could significantly advance our understanding of P. amoebophila LeuRS:

• Structural Characterization:

  • Determination of crystal structure of P. amoebophila LeuRS alone and in complex with tRNA^Leu

  • Comparison with other bacterial and eukaryotic LeuRS structures to identify unique features

  • Structure-based design of specific inhibitors

• Functional Studies:

  • Comprehensive characterization of aminoacylation and editing activities

  • Identification of tRNA recognition elements specific to this system

  • Investigation of potential moonlighting functions beyond aminoacylation

• Regulation and Expression:

  • Analysis of LeuRS expression patterns during the P. amoebophila developmental cycle

  • Investigation of post-translational modifications and their impact on activity

  • Study of potential regulatory interactions with other cellular components

• Therapeutic Applications:

  • High-throughput screening for selective inhibitors of P. amoebophila LeuRS

  • Evaluation of identified compounds for anti-Chlamydiae activity

  • Development of structure-activity relationships for lead optimization

• Systems Biology Approach:

  • Integration of LeuRS function into the broader context of P. amoebophila metabolism

  • Modeling of the impact of LeuRS inhibition on protein synthesis and bacterial viability

  • Exploration of potential synergistic drug targets in the same pathway

How might CRISPR-Cas9 techniques be applied to study P. amoebophila LeuRS function?

CRISPR-Cas9 technology offers innovative approaches to study P. amoebophila LeuRS, despite challenges associated with genetic manipulation of obligate intracellular bacteria:

• Direct Genetic Manipulation Strategies:

  • Development of transformation protocols for P. amoebophila

  • CRISPR-Cas9 delivery systems optimized for intracellular bacteria

  • Generation of conditional knockdowns or point mutations in the leuS gene

  • Creation of tagged versions for localization and interaction studies

• Host Cell Manipulation Approaches:

  • Modification of amoeba host cells to express CRISPR components

  • Targeting of host factors that interact with bacterial LeuRS

  • Creation of host cell lines expressing bacterial LeuRS to study complementation

• Cell-Free Applications:

  • In vitro CRISPR-Cas9 screening to identify functional domains

  • CRISPR interference (CRISPRi) adapted for cell-free expression systems

  • CRISPR-based biosensors for detecting LeuRS activity

• Surrogate Systems:

  • Use of genetically tractable related bacteria as models

  • Heterologous expression of P. amoebophila LeuRS in E. coli with CRISPR-Cas9 modifications

  • Complementation studies in E. coli LeuRS mutants

• Bioinformatic CRISPR Applications:

  • CRISPR-based computational screens to identify potential interaction partners

  • Prediction of guide RNA efficiency for targeting LeuRS in various experimental contexts

  • Design of CRISPR activation systems for controlled overexpression studies

While technically challenging, these approaches could provide unprecedented insights into the function and importance of LeuRS in P. amoebophila biology.

What are the essential considerations for researchers beginning work with P. amoebophila LeuRS?

Researchers initiating studies on P. amoebophila LeuRS should consider these critical factors:

• Biosafety Considerations:

  • Work with P. amoebophila requires appropriate biosafety measures (typically BSL-2)

  • The organism has been associated with pneumonia cases, highlighting potential health risks

  • Proper containment and decontamination protocols should be established

• Technical Preparation:

  • Develop expertise in amoeba culture techniques if working with the native organism

  • Establish reliable methods for recombinant protein expression and purification

  • Set up appropriate assays for aminoacylation and editing activities

  • Prepare or obtain tRNA substrates through in vitro transcription or isolation

• Collaborative Approach:

  • Consider collaborations with structural biologists for three-dimensional characterization

  • Partner with computational biologists for modeling and simulation studies

  • Engage with clinical microbiologists if pursuing pathogenesis aspects

• Resource Planning:

  • Allocate sufficient time for optimization of expression and purification

  • Budget for specialized reagents including isotopically labeled amino acids if needed

  • Plan for long-term storage of stable enzyme preparations

  • Consider throughput needs for inhibitor screening if pursuing drug development

• Research Focus Selection:

  • Clearly define whether the focus is on basic enzymology, structural biology, pathogenesis, or drug development

  • Design a logical experimental progression from basic characterization to application

  • Establish relevant comparators (e.g., human LeuRS, E. coli LeuRS) for comparative studies

What interdisciplinary approaches would be most valuable for comprehensive analysis of P. amoebophila LeuRS?

A truly comprehensive understanding of P. amoebophila LeuRS requires integration of multiple disciplines:

• Biochemistry and Molecular Biology:

  • Detailed enzymological characterization of aminoacylation and editing activities

  • Mutagenesis studies to identify critical residues

  • Protein-RNA interaction analysis

• Structural Biology:

  • X-ray crystallography or cryo-EM of the enzyme alone and in complex with substrates

  • NMR studies of dynamic aspects of enzyme function

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational changes

• Microbiology and Cell Biology:

  • Studies of LeuRS expression and localization during the developmental cycle

  • Investigation of the impact of LeuRS inhibition on bacterial viability

  • Analysis of host-pathogen interactions involving translation machinery

• Bioinformatics and Computational Biology:

  • Evolutionary analysis and phylogenetic studies

  • Molecular dynamics simulations of enzyme function

  • Systems biology modeling of the impact of LeuRS on the bacterial proteome

• Medicinal Chemistry and Pharmacology:

  • Development and optimization of selective inhibitors

  • Pharmacokinetic and pharmacodynamic studies of lead compounds

  • Toxicity assessment of potential therapeutic agents

• Clinical Microbiology:

  • Correlation of LeuRS variants with clinical outcomes

  • Development of diagnostic tools based on LeuRS detection

  • Assessment of LeuRS as a biomarker for P. amoebophila infections