Recombinant Pagrus major Estrogen receptor (esr1), partial

What is the sequence similarity of Pagrus major ESR1 compared to other species, and how does this inform structural modeling?

The predicted three-dimensional conformation of ER-α from Pagrus major (ESR1_PAGMA) demonstrates approximately 70.71% sequence similarity when compared with homologous structures in protein databases . This level of conservation provides a reliable foundation for structural modeling approaches. When comparing fish estrogen receptors, research indicates significant structural conservation within the DNA-binding domain (DBD) and ligand-binding domain (LBD), especially in species within the Sparidae family .

For researchers conducting structural studies, this conservation allows the application of homology modeling techniques using established templates. The structural analysis should focus particularly on the LBD region, as this contains the binding site for estradiol and other ligands. Methodologically, tools such as AlphaFold DB have proven effective for generating reliable structural predictions that can be further refined through molecular dynamics simulations.

How do the binding affinities of estradiol differ between ER-α and ER-β in Pagrus major, and what methodologies best capture these differences?

Docking studies reveal significant differences in binding affinities between ER subtypes in fish models. Based on comparative analysis, estradiol (E2) typically shows approximately 28% higher binding affinity with ER-β compared to ER-α . Specifically, docking simulations using AutoDock Vina demonstrate that E2 complexed with ER-β yields binding energies around -8.5 kcal/mol, while ER-α interactions with E2 show binding energies of approximately -6.6 kcal/mol .

Methodologically, researchers should employ multiple complementary approaches:

• In silico docking using AutoDock Vina or similar software

• Interaction analysis using visualization tools like LigPlot+

• Experimental validation through ligand binding assays

The binding interaction analysis reveals that while the number of polar bonds with E2 remains similar between both receptor subtypes, ER-β forms more non-polar bonds with E2, likely accounting for the stronger binding affinity . This distinction is crucial for understanding the differential response of these receptors to endocrine-disrupting compounds in marine environments.

What are the optimal expression systems for producing recombinant Pagrus major ESR1, and how do they compare to mammalian ESR1 expression systems?

Expression System Comparison:

For optimal expression in E. coli, researchers should:

• Include an N-terminal His-tag for efficient purification

• Express the protein at lower temperatures (16-18°C) to enhance solubility

• Consider expressing only the LBD or DBD separately for specific applications

• Employ buffer systems containing 20mM phosphate buffer with 150mM NaCl at pH 7.2 for optimal stability

For applications requiring full post-translational modifications, mammalian systems may be necessary despite lower yields.

What reconstitution and storage protocols maximize stability of purified recombinant Pagrus major ESR1 protein?

Proper reconstitution and storage are critical for maintaining functional integrity of recombinant ESR1 proteins. Based on established protocols for similar nuclear receptors:

Recommended Reconstitution Protocol:

• Centrifuge the lyophilized protein tube before opening to ensure all material is at the bottom

• Reconstitute to a concentration of 0.1-0.5 mg/mL using sterile distilled water

• Avoid vigorous pipetting or vortexing which can denature the protein

• For enhanced stability, add a carrier protein or stabilizer (0.1% BSA, 5% HSA, 10% FBS, or 5% Trehalose)

• Allow complete dissolution by gentle rotation at 4°C

Storage Recommendations:

• Store lyophilized protein at -20°C to -80°C for up to 1 year

• After reconstitution, the protein solution is stable at -20°C for approximately 3 months

• For short-term storage (up to 1 week), 2-8°C is acceptable

• Aliquot the reconstituted protein solution to minimize freeze-thaw cycles which significantly impact activity

Researchers should validate protein activity after reconstitution using binding assays or reporter gene assays to ensure functional integrity has been maintained.

How do mutations in the ligand-binding domain of Pagrus major ESR1 affect binding kinetics compared to those observed in human ESR1 mutations?

Mutations in the ligand-binding domain (LBD) of estrogen receptors can significantly alter binding kinetics and activation profiles. While specific data on Pagrus major ESR1 mutations is limited, comparative analysis with human ESR1 mutations provides valuable insights:

Methodological Approach for Mutation Analysis:

• Site-directed mutagenesis: Generate equivalent mutations in Pagrus major ESR1 to those observed in human ESR1 (particularly Y537S/N/C and D538G mutations which occur in the LBD)

• Binding kinetics assessment: Utilize surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine:

  • Association rates (kon)

  • Dissociation rates (koff)

  • Equilibrium dissociation constants (KD)

• Molecular dynamics simulations: Compare conformational stability between wild-type and mutant receptors

Research on human ESR1 indicates that mutations like Y537S can lead to ligand-independent activation by stabilizing the receptor in an active conformation . Similar structural analysis in Pagrus major ESR1 would determine if comparable mechanisms exist. The high conservation of the LBD suggests that mutations in equivalent positions might produce similar effects, but species-specific differences in protein dynamics may alter the magnitude of these effects.

For researchers studying these mutations, it is crucial to combine in silico modeling with experimental validation using recombinant proteins containing the mutations of interest.

What advanced computational approaches can best predict the interaction between Pagrus major ESR1 and environmental endocrine disruptors?

Environmental endocrine disruptors represent a significant concern for marine species, making predictive modeling of their interactions with estrogen receptors essential. Advanced computational approaches for studying these interactions include:

Multi-stage Computational Pipeline:

• Molecular docking simulations:

  • Primary screening using AutoDock Vina or similar tools

  • Grid box encompassing the entire ligand-binding pocket

  • Multiple docking runs (minimum 8) to ensure conformational sampling

  • Selection of poses based on lowest binding energy scores

• Interaction analysis:

  • Detailed characterization of binding poses using LigPlot+

  • Identification of key polar and non-polar interactions

  • Comparison of hydrogen bonding networks between different ligands

• Molecular dynamics (MD) simulations:

  • Minimum 100ns simulations in explicit solvent

  • Analysis of binding stability over time

  • Calculation of MM-GBSA or MM-PBSA for more accurate binding free energies

• Quantum mechanics/molecular mechanics (QM/MM):

  • For detailed electronic structure analysis of key interactions

  • Particularly valuable for compounds with unusual binding mechanisms

This multi-tiered approach allows researchers to identify potential endocrine disruptors with high confidence and understand their mechanism of action. The binding energy threshold established from estradiol docking (approximately -6.6 kcal/mol for ER-α and -8.5 kcal/mol for ER-β) provides a useful reference point for evaluating the potential potency of environmental compounds .

How does the expression profile of Pagrus major ESR1 vary across tissues and developmental stages, and what methodologies provide the most comprehensive analysis?

Understanding the spatiotemporal expression pattern of ESR1 in Pagrus major requires a combination of transcriptomic and proteomic approaches. Based on studies in related teleost species:

Recommended Methodological Approach:

• Quantitative RT-PCR:

  • Design primers specific to Pagrus major ESR1

  • Use reference genes specific to teleost fish (β-actin, EF1α, and 18S rRNA)

  • Sample collection across multiple developmental stages and tissues

  • Calculate relative expression using 2^-ΔΔCt method

• RNA-Seq analysis:

  • Deep sequencing (minimum 30M reads per sample)

  • Differential expression analysis comparing:

    • Developmental stages (embryo, larval, juvenile, adult)

    • Reproductive cycle phases

    • Different tissue types (liver, gonads, brain, etc.)

• In situ hybridization:

  • Allows spatial localization within tissues

  • Particularly valuable for brain and gonadal expression patterns

  • Use DIG-labeled RNA probes specific to ESR1

• Immunohistochemistry:

  • Requires validated antibodies against Pagrus major ESR1

  • Enables protein-level confirmation of expression patterns

Studies in related species show that ESR expression typically peaks during vitellogenesis in the liver and ovary, with a notable increase and decrease throughout the breeding cycle . This pattern reflects the receptor's critical role in reproductive physiology. For comprehensive analysis, researchers should compare expression patterns of both ER-α and ER-β, as these receptors often show complementary but distinct expression profiles.

What are the key functional differences between naturally occurring isoforms of Pagrus major ESR1, and how can they be experimentally characterized?

The functional characterization of naturally occurring isoforms of Pagrus major ESR1 requires a combination of molecular, cellular, and computational approaches. While specific isoform data for Pagrus major is limited, the methodological approach can be informed by studies of other teleost species:

Experimental Characterization Pipeline:

• Isoform identification:

  • 5' and 3' RACE to identify all expressed isoforms

  • Full-length cloning and sequencing

  • Analysis of alternative splicing patterns

• Domain structure analysis:

  • In silico prediction of functional domains using InterPro or similar tools

  • Identification of differences in critical regions (DBD, LBD, AF-1, AF-2)

  • Structural modeling of isoform-specific differences

• Reporter gene assays:

  • Transfection of each isoform construct into appropriate cell lines

  • Measurement of basal and ligand-induced transcriptional activity

  • Determination of EC50 values for estradiol and other ligands

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify isoform-specific cofactor interactions

  • Mammalian two-hybrid assays to quantify interaction strengths

  • ChIP-seq to determine genome-wide binding patterns

The functional differences between isoforms typically center on:

• Ligand sensitivity and specificity

• Cofactor recruitment profiles

• DNA binding preferences

• Subcellular localization patterns

Research in other teleost species indicates that alternative splicing often affects the AF-1 domain, resulting in isoforms with differing constitutive activities. Researchers should pay particular attention to expression patterns of these isoforms across tissues, as they may serve tissue-specific functions.

How does the three-dimensional structure of Pagrus major ESR1 compare with other teleost species, and what functional implications do these differences have?

Comparative structural analysis of Pagrus major ESR1 with other teleost species reveals important evolutionary adaptations and functional specializations. Three-dimensional structural comparisons indicate:

Structural Comparison with Related Species:

Functional Implications:

• Ligand binding properties:

  • Species-specific variations in binding pocket residues may alter ligand specificity

  • These differences can affect sensitivity to environmental estrogens

• Cofactor interactions:

  • Variations in the AF-1 and AF-2 domains likely influence cofactor recruitment

  • Species-specific cofactor interactions may explain differences in transcriptional responses

• DNA binding patterns:

  • While the DBD is highly conserved, subtle differences may alter genomic targeting

  • ChIP-seq studies would reveal species-specific binding patterns

For comprehensive functional analysis, researchers should combine structural modeling with experimental approaches such as ligand binding assays and transcriptional reporter systems using recombinant receptors from multiple species.

What evolutionary insights can be gained from comparing the Pagrus major ESR1 gene with ESR1 genes across vertebrate lineages?

Evolutionary analysis of the ESR1 gene across vertebrate lineages provides valuable insights into the functional adaptation of estrogen signaling. For Pagrus major ESR1, comparative genomic approaches reveal:

Evolutionary Analysis Methodology:

• Phylogenetic analysis:

  • Multiple sequence alignment of ESR1 coding sequences

  • Construction of maximum likelihood trees

  • Bayesian inference of evolutionary relationships

  • Dating of gene duplication events

• Selection pressure analysis:

  • Calculation of dN/dS ratios across different domains

  • Identification of sites under positive selection

  • Branch-site tests for lineage-specific selection

• Synteny analysis:

  • Examination of conserved gene neighborhoods

  • Identification of genomic rearrangements

  • Inference of ancestral genomic structure

The evolutionary trajectory of ESR1 in teleosts is particularly interesting due to the whole genome duplication event in the teleost lineage, which created additional complexity in estrogen signaling pathways. Research indicates that following this duplication, subfunctionalization and neofunctionalization of estrogen receptors occurred, leading to specialized roles for ER-α and ER-β subtypes .

In Pagrus major, as in other teleosts, the conservation of the LBD suggests strong purifying selection on ligand-binding function, while variations in other domains may reflect adaptations to specific physiological demands or environmental pressures.

For researchers interested in evolutionary aspects, combining sequence analysis with structural modeling and functional assays would provide the most comprehensive understanding of how ESR1 function has evolved across vertebrate lineages.

How can recombinant Pagrus major ESR1 be utilized in environmental monitoring for endocrine-disrupting compounds in marine ecosystems?

Recombinant Pagrus major ESR1 offers a species-relevant tool for monitoring endocrine-disrupting compounds (EDCs) in marine environments. Implementing this approach requires:

Methodological Framework for Environmental Monitoring:

• In vitro reporter assays:

  • Stable transfection of Pagrus major ESR1 into appropriate cell lines

  • Development of luciferase or other reporter systems

  • Validation with known estrogenic compounds

  • Creation of standard curves for quantitative analysis

• Competitive binding assays:

  • Development of high-throughput binding assays using recombinant ESR1

  • Fluorescence polarization or radioligand displacement methods

  • Screening of environmental samples against estradiol binding

• Biosensor development:

  • Immobilization of recombinant ESR1 on sensor surfaces

  • Integration with label-free detection systems (SPR, QCM-D)

  • Field-deployable configurations for in situ monitoring

• Validation protocol:

  • Parallel analysis with chemical methods (LC-MS/MS)

  • Deployment in gradient-exposure studies

  • Correlation with biological effects in wild populations

Using recombinant Pagrus major ESR1 provides several advantages over mammalian-based systems for marine monitoring:

• Species-relevant sensitivity to environmental contaminants

• Temperature adaptations suitable for marine environments

• Evolutionary adaptations to marine-specific EDCs

The binding characteristics determined through molecular docking studies (binding energies of approximately -6.6 kcal/mol for ER-α) provide a baseline for comparing potential EDCs . Compounds showing similar or stronger binding energies would warrant further investigation for potential endocrine-disrupting effects.

How can structural insights from Pagrus major ESR1 contribute to the design of species-specific endocrine modulators for aquaculture applications?

Structural knowledge of Pagrus major ESR1 enables rational design of species-specific endocrine modulators for potential aquaculture applications. This approach combines:

Design Strategy for Species-Specific Modulators:

• Structure-based virtual screening:

  • Use of the 3D model of Pagrus major ESR1 for in silico screening

  • Targeting of species-specific binding pocket features

  • Ranking compounds by predicted binding energy and interaction patterns

  • Selection of candidates with favorable specificity profiles

• Pharmacophore modeling:

  • Identification of key interaction features from estradiol binding

  • Development of pharmacophore models specific to Pagrus major ESR1

  • Screening of compound libraries against these models

  • Refinement based on experimental validation

• Experimental validation pipeline:

  • Binding assays with recombinant Pagrus major ESR1

  • Specificity testing against human and other vertebrate ERs

  • Cell-based transcriptional activation assays

  • Safety and efficacy testing in controlled aquaculture settings

The docking studies showing differences in binding patterns between ER-α and ER-β (particularly in non-polar interactions) provide valuable starting points for designing subtype-specific modulators . By exploiting these differences, researchers could potentially develop compounds that selectively modulate specific pathways relevant to reproduction or growth in aquaculture settings.

Importantly, all development of such modulators should include comprehensive environmental safety assessments to ensure that any compounds used in aquaculture do not adversely affect wild populations or ecosystems if released.

What strategies can overcome expression and solubility challenges when producing recombinant Pagrus major ESR1 in bacterial systems?

Researchers frequently encounter expression and solubility challenges when producing recombinant nuclear receptors like ESR1. Based on experiences with similar proteins:

Troubleshooting Strategies:

• Optimizing expression constructs:

  • Express individual domains (LBD, DBD) rather than full-length protein

  • Use solubility-enhancing tags (MBP, SUMO, Thioredoxin) in addition to His-tag

  • Optimize codon usage for E. coli expression

  • Remove flexible regions identified through disorder prediction

• Expression condition optimization:

  • Reduce induction temperature to 16-18°C

  • Use lower IPTG concentrations (0.1-0.5 mM)

  • Extend expression time (16-24 hours) at lower temperatures

  • Screen multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express)

• Solubilization strategies:

  • Include low concentrations of non-ionic detergents (0.05-0.1% Triton X-100)

  • Add stabilizing agents (5-10% glycerol, 50-100 mM arginine)

  • Optimize buffer conditions (20mM PB, 150mM NaCl, pH 7.2)

  • Consider on-column refolding protocols for inclusion bodies

• Purification optimization:

  • Implement multi-step purification (IMAC followed by ion exchange and size exclusion)

  • Include ATP wash steps to remove chaperones

  • Consider on-column refolding protocols

  • Validate proper folding through circular dichroism or limited proteolysis

Monitoring protein quality at each step using SDS-PAGE and activity assays is essential for optimizing the protocol. For the LBD, including stabilizing ligands during expression and purification can significantly improve yield and quality.

What are the critical considerations for designing accurate and reproducible ligand binding assays using recombinant Pagrus major ESR1?

Designing reliable ligand binding assays for recombinant ESR1 requires careful attention to multiple factors:

Critical Assay Design Considerations:

• Protein quality control:

  • Verify structural integrity through circular dichroism

  • Confirm monomeric status by size exclusion chromatography

  • Validate functional activity with known ligands

  • Monitor batch-to-batch consistency

• Assay format selection:

  • Radioligand binding: Highest sensitivity but requires radioactive materials

  • Fluorescence polarization: Good throughput, no separation steps

  • Surface plasmon resonance: Real-time kinetics but requires surface immobilization

  • Isothermal titration calorimetry: Direct thermodynamic parameters but lower throughput

• Assay optimization:

  • Determine optimal protein concentration (typically 10-100 nM)

  • Establish appropriate incubation times for equilibrium (1-4 hours)

  • Define optimal temperature (typically 4°C or room temperature)

  • Include appropriate controls (non-specific binding, solvent effects)

• Data analysis recommendations:

  • Use appropriate binding models (one-site, two-site, cooperative)

  • Implement global fitting for competition assays

  • Calculate Z' factor to assess assay quality

  • Perform technical and biological replicates (minimum n=3)

The docking studies indicating differential binding energies between ER subtypes (-6.6 kcal/mol for ER-α vs -8.5 kcal/mol for ER-β) suggest that assay conditions may need adjustment when working with different receptor subtypes. Researchers should validate assay parameters separately for each receptor to ensure accurate comparative studies.