Recombinant Protochlamydia amoebophila Proline--tRNA ligase (proS), partial

What is Protochlamydia amoebophila Proline--tRNA ligase and what is its function?

Protochlamydia amoebophila Proline--tRNA ligase (ProS), also known as Prolyl-tRNA synthetase (ProRS), is an essential enzyme belonging to the aminoacyl-tRNA synthetase family. This enzyme catalyzes the attachment of proline to its cognate tRNA molecules during protein synthesis, ensuring the correct incorporation of proline into nascent polypeptides. The reaction occurs through a two-step process: first, proline is activated with ATP to form prolyl-adenylate (Pro-AMP), followed by the transfer of activated proline to the 3'-end of tRNA^Pro.

As a member of the class II aminoacyl-tRNA synthetases, P. amoebophila ProRS likely contains pre- and post-editing mechanisms to prevent mischarging of tRNA with similar amino acids, thereby maintaining translational fidelity. These editing mechanisms are critical for preventing mistranslation that could lead to protein misfolding and cellular dysfunction. Bacterial ProRS contains both pre-transfer editing, where mis-activated amino acid adenylates are hydrolyzed before attachment to tRNA, and post-transfer editing, where non-cognate amino acids attached to tRNA^Pro are hydrolyzed .

In the context of P. amoebophila, an obligate intracellular bacterial symbiont of amoebae within the Chlamydiae phylum, ProRS represents not only an essential component of its protein synthesis machinery but also a potential target for studying the evolution of aminoacyl-tRNA synthetases in organisms adapted to intracellular lifestyles. Understanding the structure and function of this enzyme provides insights into both fundamental biology and potential therapeutic interventions.

How is recombinant P. amoebophila ProRS typically expressed and purified?

The expression and purification of recombinant P. amoebophila ProRS typically follows protocols established for other bacterial ProRS enzymes, with specific optimizations to address solubility challenges. Based on methodologies used for related enzymes, the following approach is recommended:

Expression System Setup:

• The proS gene from P. amoebophila genomic DNA should be PCR-amplified, cloned into an expression vector (typically pET-based), and transformed into an E. coli expression strain such as BL21(DE3).

• Expression conditions require careful optimization, particularly regarding temperature and inducer concentration. For instance, P. aeruginosa ProRS was optimally expressed at 30°C with 25 μM IPTG to minimize inclusion body formation .

• Pilot expressions should test multiple conditions, including lower temperatures (16-30°C), IPTG concentrations (25-100 μM), and induction times (4-24 hours) to maximize soluble protein yield.

Purification Protocol:

• Cell lysis using sonication or mechanical disruption in a buffer containing protease inhibitors

• Clarification of lysate by high-speed centrifugation (typically 20,000-40,000 × g for 30-60 minutes)

• Initial purification via affinity chromatography (Ni-NTA if His-tagged)

• Secondary purification through ion-exchange chromatography (typically Q or SP Sepharose)

• Final polishing step using size-exclusion chromatography

• Buffer exchange to a storage buffer containing reducing agents and glycerol

Optimization is critical, as P. aeruginosa ProRS required specific conditions to overcome initial solubility issues. The expression was considerably improved by reducing both growth temperature and IPTG concentration . The purity level should be assessed by SDS-PAGE and should exceed 95% for enzymatic studies, as was achieved with P. aeruginosa ProRS .

Additionally, the storage buffer should typically contain DTT or β-mercaptoethanol to maintain reduced cysteines, and activity should be verified through aminoacylation assays using purified or in vitro transcribed tRNA^Pro. This approach ensures the production of functionally active enzyme suitable for biochemical and structural characterization.

What are the key structural domains of P. amoebophila ProRS?

While the specific structure of P. amoebophila ProRS has not been fully characterized in published literature, its key structural domains can be inferred from homologous ProRS enzymes from other bacterial species. As a class II aminoacyl-tRNA synthetase, P. amoebophila ProRS likely contains the following domains and structural features:

Catalytic Domain:

• Contains the ATP binding site with highly conserved class II motifs, including the HIGH and KMSKS motifs

• Houses the proline binding pocket with conserved residues that specifically recognize the unique cyclic structure of proline

• Likely includes residues analogous to those in P. aeruginosa ProRS, where the imino group of proline forms hydrogen bonds with conserved threonine and glutamate residues (Thr109 and Glu111 in E. faecalis numbering), while the carbonyl-oxygen interacts with a conserved arginine residue (Arg140)

• Features a characteristic antiparallel β-sheet structure surrounded by α-helices, typical of class II aminoacyl-tRNA synthetases

Anticodon Binding Domain:

• Responsible for recognizing the anticodon loop of tRNA^Pro

• Contains specific residues that interact with the unique features of tRNA^Pro

• Ensures specificity for the correct tRNA isoacceptor

Editing Domain:

• May contain both pre- and post-transfer editing sites to hydrolyze mischarged aminoacyl-adenylate or mischarged tRNA

• These mechanisms prevent misincorporation of alanine, cysteine, or other near-cognate amino acids during protein synthesis

Structural analysis of related bacterial ProRS enzymes reveals that key active site residues are highly conserved, suggesting that P. amoebophila ProRS would maintain similar structural features while potentially exhibiting adaptations specific to its intracellular lifestyle within amoeba hosts.

How does P. amoebophila ProRS compare to ProRS enzymes from other bacterial species?

P. amoebophila ProRS, as an enzyme from an obligate intracellular bacterium within the Chlamydiae phylum, likely shares significant structural and functional similarities with ProRS enzymes from other bacterial species while exhibiting unique adaptations related to its specialized lifestyle. Comparative analysis reveals:

Sequence and Structural Conservation:

Enzymatic Mechanisms:

• Likely maintains both pre- and post-transfer editing mechanisms described for bacterial ProRS enzymes

• Two-step aminoacylation reaction (activation followed by transfer) consistent with other bacterial ProRS enzymes

Evolutionary Adaptations:

• Potential streamlining of certain domains due to genome reduction common in obligate intracellular bacteria

• Possible modifications in surface properties reflecting adaptation to the intracellular environment of amoeba hosts

• The enzyme likely retains high conservation in functional regions while showing greater variability in non-catalytic sections

Chlamydial-Specific Features:

• As a member of the Chlamydiae phylum, P. amoebophila ProRS may reflect evolutionary patterns specific to this group of bacteria

• Approximately 5-8% of Chlamydia spp. coding sequences are estimated to be effectors , but ProRS, being a core metabolic enzyme, likely shows higher conservation across species

ATP-Binding Site Architecture:

• The ATP-binding pocket of P. amoebophila ProRS may share similarities with the characterized plasticity observed in T. gondii PRS, which accommodates various inhibitors through an induced-fit mechanism

• This region could be a target for species-selective inhibitors, as demonstrated in T. gondii studies

Comparing P. amoebophila ProRS with enzymes from free-living bacteria and other intracellular pathogens provides insights into how essential enzymes adapt to different ecological niches while maintaining their fundamental catalytic functions. These comparisons also highlight potential targets for selective inhibition that could exploit structural differences between bacterial and human enzymes.

What are the typical kinetic parameters of P. amoebophila ProRS?

While specific kinetic parameters for P. amoebophila ProRS have not been extensively documented in published literature, we can infer likely parameters based on well-characterized ProRS enzymes from related bacterial species. The kinetic profile of P. aeruginosa ProRS provides a useful reference point:

Substrate Affinities (KM values):

• ATP: 154 μM

• Proline: 122 μM

• tRNA^Pro: 5.5 μM

Catalytic Efficiency:

• The turnover number (kcat) for P. aeruginosa ProRS with tRNA^Pro was determined to be approximately 0.2 s^-1

• The catalytic efficiency (kcat/KM) for tRNA^Pro was calculated to be 0.035 s^-1 μM^-1

Experimental Methodology for Determining Kinetic Parameters:

To determine the kinetic parameters for P. amoebophila ProRS, researchers would typically employ the following approaches:

• ATP Kinetics:

  • Vary ATP concentration (typically 25-1000 μM) while maintaining fixed saturating concentrations of proline and tRNA^Pro

  • Measure initial velocities and fit to the Michaelis-Menten equation to determine KM and kcat values

  • Similar to studies with P. aeruginosa ProRS, where ATP concentrations between 25-1000 μM were used to determine KM

• Proline Kinetics:

  • Vary proline concentration (typically 10-500 μM) with fixed saturating ATP and tRNA^Pro

  • Plot initial velocities versus proline concentration to determine KM and kcat

• tRNA^Pro Kinetics:

  • Vary tRNA^Pro concentration (typically 0.5-5 μM) while keeping ATP and proline at saturating levels

  • Similar to the approach used with P. aeruginosa ProRS, where tRNA^Pro concentrations between 0.75-3.0 μM were used

The aminoacylation reaction would be monitored using established methods such as the incorporation of radioactively labeled proline into tRNA, followed by acid precipitation and scintillation counting. The initial velocities would be modeled by fitting them to the Michaelis-Menten steady-state model, as was done for P. aeruginosa ProRS .

These kinetic parameters are influenced by reaction conditions including pH (typically 7.0-8.0), temperature (typically 25-37°C), salt concentration (50-200 mM KCl or NaCl), and the presence of divalent cations (particularly Mg²⁺ at 5-10 mM). Optimization of these conditions specifically for P. amoebophila ProRS would be necessary for accurate kinetic characterization.

What strategies can be employed to improve the solubility of recombinant P. amoebophila ProRS during expression?

Recombinant expression of aminoacyl-tRNA synthetases, including ProRS from various species, often presents solubility challenges that require systematic optimization. For P. amoebophila ProRS, multiple strategies can be employed to enhance solubility:

Expression Parameter Optimization:

• Temperature reduction during induction is critical, as demonstrated for P. aeruginosa ProRS where a 30°C induction temperature was optimal . Further reduction to 16-25°C may be necessary for P. amoebophila ProRS.

• IPTG concentration titration is essential, with P. aeruginosa ProRS showing optimal expression at just 25 μM IPTG . A concentration range of 10-100 μM should be tested to find the optimal balance between expression level and solubility.

• Induction time optimization, with extended expression periods (16-24 hours) at lower temperatures often yielding more soluble protein.

• Media formulation adjustments, including the use of specialty media like Terrific Broth or auto-induction media, which can significantly improve soluble protein yields.

Genetic Engineering Approaches:

• Fusion with solubility-enhancing tags represents a powerful strategy. The maltose-binding protein (MBP) tag is particularly effective for improving solubility while maintaining enzymatic activity.

• Codon optimization for E. coli expression can address potential rare codon usage in P. amoebophila genes that might lead to translational pausing and protein misfolding.

• Domain-based expression strategies may be employed if the full-length protein proves recalcitrant to soluble expression. The catalytic domain can often be expressed more readily than the complete protein.

• Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) can significantly enhance proper folding and solubility, particularly for complex multi-domain proteins like ProRS.

Buffer and Additive Optimization:

• Inclusion of osmolytes such as glycerol (5-15%), sucrose (5-10%), or arginine (50-200 mM) in lysis and purification buffers can significantly improve solubility.

• Salt concentration optimization (typically 100-500 mM NaCl) to shield charge-charge interactions that might promote aggregation.

• Reducing agents (5-10 mM DTT or β-mercaptoethanol) to prevent non-native disulfide bond formation.

• Low concentrations of non-ionic detergents (0.05-0.1% Triton X-100 or 0.5-1% CHAPS) can help solubilize proteins with exposed hydrophobic regions.

Alternative Expression Systems:

• Baculovirus-insect cell expression often provides superior folding for complex proteins that are challenging to express in bacteria.

• Cell-free protein synthesis systems allow precise control of the reaction environment and can be optimized for soluble expression.

• Cold-adapted E. coli strains like Arctic Express, which contain cold-adapted chaperonins, can facilitate proper folding at lower temperatures.

When expressing P. aeruginosa ProRS, researchers observed that "expression of ProRS initially resulted in substantial amounts of insoluble protein," but this was overcome by "optimizing expression at various temperatures and various concentrations of IPTG" . A similar systematic optimization approach for P. amoebophila ProRS would likely yield improvements in soluble protein production.

How can the aminoacylation activity of P. amoebophila ProRS be optimized for in vitro studies?

Optimizing the aminoacylation activity of P. amoebophila ProRS for in vitro studies requires systematic adjustment of multiple parameters affecting enzyme function. A comprehensive optimization strategy should address:

Buffer Composition Optimization:

• pH screening is critical, typically testing the range of pH 7.0-8.0 in 0.2-0.3 pH unit increments

• Buffer type selection comparing HEPES, Tris, and phosphate buffers (typically 50-100 mM) for optimal enzyme activity

• Ionic strength adjustment, typically testing 50-200 mM KCl or NaCl, as ionic strength affects both enzyme stability and substrate interactions

• Divalent cation optimization, particularly Mg²⁺ (typically 5-20 mM), which is essential for ATP binding and catalysis

• Addition of stabilizing agents such as glycerol (5-20%) to maintain enzyme stability during extended reactions

Substrate Preparation and Quality:

• Production of high-quality tRNA^Pro substrate either through in vitro transcription or purification from overexpressing cells

• Verification of tRNA folding through thermal denaturation and renaturation protocols

• Titration of substrate concentrations to determine optimal ranges (ATP: typically 1-5 mM; proline: typically 0.1-1 mM; tRNA^Pro: typically 1-10 μM)

• Assessment of substrate quality through analytical methods (HPLC for nucleotides, mass spectrometry for amino acids)

Reaction Conditions:

• Temperature optimization between 25-37°C, with temperature stability monitoring

• Enzyme concentration titration to ensure measurements are made in the linear range of activity

• Incubation time optimization to ensure measurements are taken within the initial, linear phase of the reaction

• DMSO tolerance assessment if inhibitor studies are planned, as P. aeruginosa ProRS maintained significant activity with up to 10% DMSO

Optimization Process Implementation:

• Initial conditions based on P. aeruginosa ProRS (100 mM HEPES pH 7.5, 10 mM MgCl₂, 50 mM KCl, 1 mM DTT, 2 mM ATP, 100 μM L-proline, 2 μM tRNA^Pro)

• One-factor-at-a-time optimization of critical parameters

• Factorial design for multi-parameter optimization of interacting factors

• Validation through determination of Michaelis-Menten parameters

• Reproducibility testing across multiple enzyme preparations

Detection Method Selection and Optimization:

• Radioactive assays using [³H] or [¹⁴C]-proline provide high sensitivity but require appropriate safety measures

• Filter-binding assays for tRNA aminoacylation must be optimized for washing conditions and filter type

• If developing a high-throughput screening assay, parameters such as Z' factor should be determined to ensure assay robustness

P. aeruginosa ProRS aminoacylation activity was successfully monitored through a carefully optimized assay that maintained linearity with respect to time and enzyme concentration . Similar optimization for P. amoebophila ProRS would involve systematic screening of conditions, potentially employing design of experiments (DOE) approaches to efficiently identify optimal parameters for maximum enzyme activity.

What editing mechanisms does P. amoebophila ProRS employ to ensure fidelity?

Aminoacyl-tRNA synthetases, including ProRS enzymes, have evolved sophisticated editing mechanisms to maintain translational fidelity by preventing mischarging of tRNAs with incorrect amino acids. While specific information about P. amoebophila ProRS editing is not extensively documented, we can infer likely mechanisms based on bacterial ProRS enzymes:

Pre-transfer Editing Mechanisms:

• Hydrolysis of mis-activated aminoacyl-adenylate (aa-AMP) intermediates before transfer to tRNA

• According to studies on bacterial ProRS, "the pre-transfer editing occurs...in which the mis-activated amino acid (adenylate) is hydrolyzed before attachment to the 3′-end of tRNA^Pro"

• This mechanism likely discriminates against near-cognate amino acids such as alanine and cysteine that might form unstable adenylates

• May occur within the synthetic active site or at a specialized editing site within the enzyme

Post-transfer Editing Mechanisms:

• Hydrolysis of mischarged tRNA after amino acid attachment to the 3'-end of tRNA

• As described for bacterial ProRS, "the post-transfer state in which the non-cognate amino acid of the mischarged tRNA^Pro is hydrolyzed"

• Requires specific architecture to position the 3' end of the mischarged tRNA in the editing active site

• May involve conformational changes in the enzyme-tRNA complex

Research Approaches to Characterize Editing Mechanisms:

How can site-directed mutagenesis be used to study the active site of P. amoebophila ProRS?

Site-directed mutagenesis represents a powerful approach to investigate structure-function relationships within the active site of P. amoebophila ProRS. A comprehensive mutagenesis strategy would target key functional regions based on structural insights from related ProRS enzymes:

Strategic Target Selection for Mutagenesis:

• ATP-binding Pocket Residues:

  • Conserved residues in the HIGH motif essential for ATP binding and activation

  • Residues within the KMSKS motif that position ATP and participate in catalysis

  • Residues forming the ATP-binding pocket analogous to the 470-483 region in T. gondii PRS, which shows "remarkable plasticity in the ATP pocket...to accommodate these PPL-derivatives"

  • Residues that interact with inhibitors, such as those equivalent to Thr482 in T. gondii, where "the 6-methylpyridine N atom interacts with the main-chain N atom of Thr482"

• Proline-binding Pocket Residues:

  • Conserved residues involved in proline recognition, analogous to Thr109, Glu111, and Arg140 (E. faecalis numbering) where "the imino group of proline is hydrogen bonded to Thr109 and Glu111 while the carbonyl-oxygen interacts with Arg140"

  • Residues that confer specificity for proline over near-cognate amino acids

  • Residues forming the binding pocket that differentiate bacterial from human enzymes

• tRNA Interaction Sites:

  • Residues in the anticodon binding domain that recognize tRNA^Pro

  • Residues contacting the acceptor stem and positioning the 3' end for aminoacylation

  • Residues involved in conformational changes during tRNA binding

• Editing Site Residues:

  • Amino acids potentially involved in pre-transfer editing of mis-activated adenylates

  • Residues participating in post-transfer editing of mischarged tRNA

  • Residues that discriminate between cognate and non-cognate amino acids

Mutagenesis Design Strategies:

• Alanine-scanning Mutagenesis:

  • Systematic replacement of targeted residues with alanine to eliminate side chain interactions while maintaining backbone structure

  • Identification of residues essential for substrate binding versus catalysis

• Conservative Substitutions:

  • Replacing residues with chemically similar amino acids to probe specific contributions

  • Example: Thr→Ser to maintain hydroxyl functionality while reducing side chain size

• Charge-altering Mutations:

  • Reversing or neutralizing charges in electrostatic interactions

  • Example: Arg→Glu to assess the importance of positive charge in substrate binding

• Catalytic Residue Mutations:

  • Targeting residues directly involved in chemistry

  • Example: Glu→Gln to eliminate carboxylate catalytic function while maintaining steric requirements

Functional Analysis of Mutants:

• Steady-state Kinetics:

  • Determination of KM and kcat for ATP, proline, and tRNA^Pro for each mutant

  • Calculation of catalytic efficiency (kcat/KM) to quantify effects on catalysis versus binding

• Substrate Binding Studies:

  • Isothermal titration calorimetry to measure substrate binding affinities directly

  • Thermal shift assays to assess the impact of mutations on protein stability and ligand binding

• Pre-steady-state Kinetics:

  • Analysis of individual reaction steps to identify rate-limiting processes affected by mutations

  • Determination of chemical versus physical steps in the reaction pathway

• Inhibitor Sensitivity Analysis:

  • Testing mutant sensitivity to known ProRS inhibitors

  • Identification of resistance mutations similar to the T477A and T592S mutations in T. gondii PRS that "substantially reduced susceptibility to L35 compared to wild-type parasites"

Site-directed mutagenesis studies of P. amoebophila ProRS would provide valuable insights into its catalytic mechanism, substrate specificity, and potential as a drug target, while also contributing to our understanding of how this essential enzyme has adapted to the specialized intracellular environment of its amoeba host.

What approaches can be used to identify inhibitors of P. amoebophila ProRS?

Identifying selective inhibitors of P. amoebophila ProRS requires a multi-faceted approach combining computational, biochemical, and structural methods. A comprehensive inhibitor discovery strategy would include:

High-throughput Screening (HTS) Approaches:

Structure-based Approaches:

• Homology Modeling:

  • Development of P. amoebophila ProRS structural models based on related crystal structures

  • Refinement through molecular dynamics simulations

  • Validation using experimental data

• Virtual Screening:

  • Molecular docking against ATP-binding and proline-binding pockets

  • Pharmacophore-based screening using key interaction features

  • Targeting regions with potential selectivity compared to human ProRS

• Structure-guided Design:

  • Fragment-based approaches starting from validated binding fragments

  • Structure-activity relationship development based on docking poses

  • Design of compounds targeting specific binding pockets

ATP-mimetic Compounds:

• PPL Derivative Development:

  • Focus on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffolds that showed activity against T. gondii PRS

  • Targeting the ATP-binding pocket, which in T. gondii PRS "accommodates these PPL-derivatives in a groove forming the binding pocket that is composed of Arg470, Glu472, Lys474, Arg481, Thr482"

  • Structural modifications guided by structure-activity relationships observed in the T. gondii studies

• Selectivity Optimization:

  • Focus on regions that differ between bacterial and human ProRS

  • Development of compounds that exploit the "ligand-induced fit model between the ATP pocket (470-483) and the loop (528-538)"

Hit Validation and Characterization:

• Dose-response Profiling:

  • Determination of IC₅₀ values against P. amoebophila ProRS

  • Counter-screening against human ProRS for selectivity assessment

• Mechanism of Action Studies:

  • Kinetic analysis to determine competitive, noncompetitive, or uncompetitive inhibition

  • Direct binding studies using biophysical methods (ITC, SPR, thermal shift)

• Resistance Mechanism Investigation:

  • Generation of resistant mutants through in vitro selection

  • Identification of resistance mutations, similar to the approach in T. gondii where "cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen"

  • Structural analysis of resistance mechanisms

Development of P. amoebophila ProRS inhibitors would benefit from the experience with T. gondii PRS, where "five compounds centered on the PPL scaffolds were investigated for their therapeutic efficacy in killing both the Toxoplasma parasites and the Toxoplasma encoded PRS enzyme" . This approach could provide valuable starting points for inhibitor development targeting P. amoebophila ProRS.