Recombinant Ashbya gossypii FK506-binding protein 1 (FPR1)

What is Ashbya gossypii FK506-binding protein 1 (FPR1) and what is its biological role?

Ashbya gossypii FK506-binding protein 1 (FPR1) is a gene that encodes a peptidyl-prolyl cis-trans isomerase (PPIase) belonging to the immunophilin family. The protein product (FKBP12) has a conserved structure comprising five to six β-sheets that wrap around a central α-helix, with three extended loops (40s, 50s, and 80s loops) surrounding the binding pocket. In filamentous fungi like A. gossypii, FKBP12 serves as the cellular receptor for immunosuppressive drugs such as FK506 (tacrolimus) .

The biological role of FKBP12 in A. gossypii includes protein folding, signal transduction, and possibly involvement in self-substrate recognition through dimerization. The protein can form dimers through a unique interaction where the 80s loop from one monomer docks into the active site of the second monomer, a characteristic observed in other filamentous fungi such as Aspergillus fumigatus .

How does A. gossypii FKBP12 compare structurally with FKBP12 from other fungi and humans?

A. gossypii FKBP12 shares significant structural homology with FKBP12 from other fungi and humans, but with notable differences that influence drug binding and protein-protein interactions:

Based on homology with other fungal FKBP12 proteins, A. gossypii FKBP12 likely contains critical residues that distinguish it from human FKBP12, particularly in the 40s and 80s loops, which affect drug binding and protein-protein interactions .

How is the FPR1 gene regulated in A. gossypii under normal and stress conditions?

When A. gossypii is subjected to secretion stress (induced by DTT), it experiences substantial transcriptional changes. Unlike the conventional unfolded protein response (UPR) observed in other fungi, A. gossypii displays alternative protein quality control mechanisms. This includes up-regulation of genes involved in protein unfolding, endoplasmic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, and mRNA degradation .

What are the most effective protocols for cloning and expressing recombinant A. gossypii FPR1 in heterologous systems?

For efficient cloning and expression of recombinant A. gossypii FPR1, researchers can follow this optimized protocol:

Cloning Strategy:

• Amplify the A. gossypii FPR1 gene from genomic DNA or cDNA using high-fidelity PCR.

• Design primers with appropriate restriction sites or overhangs for downstream cloning.

• Consider using CRISPR/Cas9 for precise manipulation of the FPR1 gene within the A. gossypii genome itself .

Expression Systems:

• For structural studies: E. coli BL21(DE3) with pET or pGEX vectors (GST-tagged or His-tagged constructs)

• For functional studies: S. cerevisiae expression systems may be preferable due to phylogenetic proximity to A. gossypii

Expression Optimization:

• Induce expression at lower temperatures (16-20°C) to enhance protein folding

• Consider codon optimization for the expression host

• Include a cleavable tag (His, GST, or MBP) to facilitate purification while allowing tag removal

Purification Protocol:

• Cell lysis using sonication or French press in buffer containing protease inhibitors

• Affinity chromatography using the appropriate resin for the tag

• Tag cleavage if necessary

• Size exclusion chromatography for final purification

While these recommendations are inferred from general recombinant protein techniques and knowledge of A. gossypii's phylogenetic relationship to yeast, specific optimization may be required for the A. gossypii FPR1 protein .

How can I use CRISPR/Cas9 to engineer modifications in the FPR1 gene of A. gossypii?

The CRISPR/Cas9 system has been adapted for A. gossypii genome editing and can be effectively used to modify the FPR1 gene:

CRISPR/Cas9 Protocol for A. gossypii FPR1 Modification:

• Design of guide RNA (gRNA):

  • Identify target sequences within the FPR1 gene with a 5′-NGG-3′ PAM sequence

  • Design gRNA with 20 nucleotides complementary to the target sequence

  • Verify specificity using bioinformatic tools to avoid off-target effects

• Construction of CRISPR/Cas9 Vector:

  • Use the one-vector CRISPR/Cas9 system adapted for A. gossypii

  • Clone the gRNA expression cassette into the vector containing Cas9

  • If making specific mutations, include a repair template with desired modifications

• Transformation and Selection:

  • Transform A. gossypii spores with the CRISPR/Cas9 vector

  • Select transformants using appropriate markers

  • Verify genome modifications by PCR and sequencing

• Marker-free Engineering (if desired):

  • The one-vector system allows for marker-free engineering strategies

  • Design the repair template to include desired mutations without selection markers

  • Verify modifications through phenotypic or molecular screening

This approach allows for precise manipulation of the FPR1 gene, including point mutations to study structure-function relationships, gene deletions to assess essentiality, or tagging for localization or purification studies .

What methodologies are recommended for assessing the peptidyl-prolyl isomerase activity of recombinant A. gossypii FKBP12?

For assessing the peptidyl-prolyl isomerase (PPIase) activity of recombinant A. gossypii FKBP12, several complementary methodologies are recommended:

Spectrophotometric Chymotrypsin-Coupled Assay:

• Use synthetic tetrapeptide substrates containing proline (e.g., Suc-Ala-Leu-Pro-Phe-pNA)

• Monitor cis-to-trans isomerization by measuring the release of p-nitroaniline at 390 nm

• Conduct assays with and without FK506 to assess inhibition

• Calculate kinetic parameters (kcat/KM) to quantify catalytic efficiency

Protease-Free NMR-Based Assay:

• Use 15N-labeled peptide substrates

• Monitor cis/trans isomerization by 2D 1H-15N HSQC NMR spectroscopy

• Calculate isomerization rates from time-dependent spectral changes

• This technique provides direct measurement without protease coupling

FK506 Binding Assays:

• Isothermal titration calorimetry (ITC) to determine binding affinities (KD)

• Thermal shift assays to assess protein stability upon ligand binding

• Surface plasmon resonance (SPR) for real-time binding kinetics

Comparative Analysis:
Compare the activity of A. gossypii FKBP12 with that of other fungal and human FKBP12 proteins using the same substrates and conditions to identify species-specific differences in catalytic efficiency and inhibitor sensitivity.

These methodologies enable comprehensive characterization of both the enzymatic activity and drug-binding properties of A. gossypii FKBP12, providing insights into its functional properties.

How do mutations in the 40s, 50s, and 80s loops of A. gossypii FKBP12 affect its interaction with FK506 and calcineurin?

Based on studies with other fungal FKBP12 proteins, mutations in the 40s, 50s, and 80s loops of A. gossypii FKBP12 would likely have significant effects on its interactions with FK506 and calcineurin:

40s Loop Mutations:

• F22T, Q50M, and R55E substitutions (corresponding to A. fumigatus positions) would likely impact FK506-FKBP12 interactions with calcineurin

• In A. fumigatus, mutations in the 40s loop (F37M/L) decreased calcineurin binding and increased resistance to FK506

50s Loop Mutations:

• W60V mutation (based on A. fumigatus) would likely decrease calcineurin binding

• This mutation in A. fumigatus increased contacts in the 80s loop, resulting in higher resistance to FK506

80s Loop Mutations:

• The 80s loop is particularly critical as it contains a proline residue involved in FKBP12 dimerization

• F88H mutation (corresponding to the difference between fungal and human FKBP12) would likely result in significant increase in resistance to FK506 due to reduced binding of FK506-FKBP12 complex with calcineurin

• P90G (or equivalent position in A. gossypii) would likely disrupt dimerization and alter FK506 binding

These structure-function relationships highlight potential targets for developing antifungal compounds with specificity for fungal FKBP12 while minimizing interaction with human FKBP12.

What are the key differences in FK506 binding between A. gossypii FKBP12 and human FKBP12, and how can these be exploited for drug development?

The key differences in FK506 binding between A. gossypii FKBP12 (inferred from other fungal FKBP12 proteins) and human FKBP12 present opportunities for selective drug development:

Critical Structural Differences:

Exploitation for Drug Development:

• Design FK506 analogs that interact preferentially with F88 in fungal FKBP12 rather than H88 in human FKBP12

• Develop compounds that target the unique dimerization interface of fungal FKBP12

• Create modified compounds like L685,818 (a C18 hydroxy, C21 ethyl derivative of FK506) that maintain antifungal activity while reducing immunosuppressive effects

• Design molecules that exploit differences in the 40s loop residues to enhance selectivity

These structural differences provide a foundation for developing non-immunosuppressive antifungal agents that selectively target fungal FKBP12 while minimizing interaction with human FKBP12 .

How does dimerization of A. gossypii FKBP12 influence its biological activity and drug interactions?

Dimerization of A. gossypii FKBP12, inferred from studies on other fungal FKBP12 proteins, has significant implications for its biological activity and drug interactions:

Mechanism of Dimerization:

• In fungal FKBP12 proteins, the 80s loop from one monomer docks into the active site of the second monomer and vice-versa

• A specific proline residue at the tip of the 80s loop (P90 in A. fumigatus or equivalent in A. gossypii) facilitates this dimerization

• This dimerization appears to be unique to fungal FKBP12 and is not observed in human FKBP12

Functional Implications:

• Auto-regulation: Dimerization may serve as an auto-regulatory mechanism by blocking the active site

• Drug competition: The self-substrate region overlaps with the FK506 binding region, meaning FK506 must compete with dimerization

• Resistance mechanism: Mutations affecting dimerization (such as P90G in A. fumigatus) confer resistance to FK506, indicating a relationship between dimerization and drug binding

• Species-specific differences: In C. albicans, all FKBP12 proteins have P104 in cis conformation, while in A. fumigatus, the proline can be in either cis or trans states, suggesting species-specific dimerization dynamics

Implications for Drug Design:

• Compounds that specifically disrupt fungal FKBP12 dimerization might represent a novel class of antifungals

• Understanding the dynamic between dimerization and drug binding could inform the design of more potent inhibitors

• The species-specific differences in dimerization dynamics could potentially be exploited for selective targeting of particular fungal pathogens

These insights highlight dimerization as a unique feature of fungal FKBP12 proteins that has significant implications for drug binding and could be exploited for selective antifungal development .

How does the A. gossypii FKBP12-FK506-calcineurin complex differ from the human complex, and what are the implications for antifungal selectivity?

The A. gossypii FKBP12-FK506-calcineurin complex likely exhibits important differences from the human complex based on findings from other fungal species:

Structural Differences:

Implications for Antifungal Selectivity:

• FK506 Analogs:

  • L685,818, a C18 hydroxy, C21 ethyl derivative of FK506, demonstrates a key principle: while L685,818-human FKBP12 cannot inhibit calcineurin, L685,818-fungal FKBP12 can still inhibit calcineurin

  • This differential effect provides a foundation for developing non-immunosuppressive antifungals

• Exploitable Differences:

  • The F88 vs. H88 difference presents a clear target for selective inhibitor design

  • Variations in the 40s loop (F22 vs. T22, Q50 vs. M50, R55 vs. E55) provide additional targets for selective binding

  • Compounds could be designed to take advantage of the unique dimerization properties of fungal FKBP12

• Cross-Species Considerations:

  • Even among fungi, there are species-specific differences (e.g., McFKBP12 has Y88 instead of F88, affecting FK506 binding)

  • These differences could be exploited for developing species-specific antifungals

These structural and functional differences provide a foundation for developing antifungal compounds that selectively target fungal FKBP12-calcineurin interactions while minimizing effects on the human pathway.

What experimental approaches can determine if A. gossypii calcineurin is essential for growth, and how does this compare to other fungi?

To determine if calcineurin is essential for growth in A. gossypii and compare with other fungi, several experimental approaches can be used:

Genetic Approaches:

• CRISPR/Cas9 Gene Disruption:

  • Use the one-vector CRISPR/Cas9 system adapted for A. gossypii to target the calcineurin catalytic (CNA) or regulatory (CNB) subunit genes

  • Analyze the resulting phenotypes under various growth conditions

  • If mutants cannot be recovered, it suggests essentiality

• Conditional Expression Systems:

  • Generate strains with calcineurin genes under control of inducible/repressible promoters

  • Monitor growth under repressing conditions

  • This approach is valuable if calcineurin is essential, as it allows controlled depletion

Pharmacological Approaches:

• FK506 Sensitivity Assays:

  • Determine minimum inhibitory concentrations (MICs) of FK506 for A. gossypii

  • Compare with other fungi to assess relative sensitivity

  • Test growth on solid and liquid media with varying FK506 concentrations

• Cyclosporin A Studies:

  • As an alternative calcineurin inhibitor, compare effects of cyclosporin A with FK506

  • Differential sensitivity may reveal aspects of pathway specificity

Functional Genomics:

• Transcriptomic Analysis:

  • Compare gene expression profiles of A. gossypii under calcineurin inhibition (using DTT or FK506)

  • Identify calcineurin-dependent genes and pathways

  • In A. gossypii treated with DTT, widespread transcriptional changes occur that may include calcineurin pathway components

Comparative Analysis:

• In many pathogenic fungi, calcineurin is not essential for viability but is crucial for virulence and stress responses

• A. gossypii may differ from pathogenic fungi due to its saprophytic lifestyle

• Compare findings to calcineurin dependency in related species like S. cerevisiae, which is phylogenetically close to A. gossypii

These approaches would provide comprehensive insights into the role of calcineurin in A. gossypii growth and development, informing potential antifungal strategies targeting this pathway.

How do non-immunosuppressive FK506 analogs interact with A. gossypii FKBP12, and what is their antifungal efficacy?

Non-immunosuppressive FK506 analogs would interact with A. gossypii FKBP12 in ways that exploit the structural differences between fungal and human FKBP12-calcineurin interactions:

FK506 Analog Interactions:

Antifungal Efficacy:

Factors Affecting Efficacy:

• Species-Specific Differences:

  • Even among fungi, variations in the FKBP12 sequence can affect drug binding

  • For example, Mucor circinelloides FKBP12 has Y88 instead of F88, which affects FK506 binding

  • These differences highlight the need for careful consideration of the target fungal species

• Resistance Mechanisms:

  • Mutations in the 80s loop (e.g., P90G in A. fumigatus) confer FK506 resistance

  • Monitoring for such mutations in A. gossypii would be important for clinical applications

  • Understanding the resistance mechanism allows for the design of second-generation inhibitors

These insights provide a foundation for developing antifungal agents based on FK506 that retain activity against A. gossypii while minimizing immunosuppressive effects in humans .

What are the key technical challenges in expressing and purifying sufficient quantities of A. gossypii FKBP12 for structural studies?

Researchers face several technical challenges when expressing and purifying A. gossypii FKBP12 for structural studies:

Expression Challenges:

• Codon Usage Optimization:

  • A. gossypii has a different codon bias than common expression hosts

  • Codon optimization may be necessary for efficient expression in E. coli or yeast systems

  • Failure to optimize can lead to truncated protein products or inclusion body formation

• Protein Folding:

  • As a PPIase, FKBP12 has specific folding requirements

  • Misfolding in heterologous systems may occur, especially at high expression levels

  • Expression at lower temperatures (16-20°C) and slower induction rates may improve folding

• Expression System Selection:

  • E. coli systems may lack appropriate post-translational modifications

  • Yeast systems might be more suitable given that A. gossypii is phylogenetically closer to yeast

  • Insect cell systems may provide a compromise between yield and proper folding

Purification Challenges:

• Solubility Issues:

  • Fungal FKBP12 proteins can form dimers through the 80s loop

  • This dimerization may affect solubility and homogeneity

  • Addition of FK506 during purification might prevent dimerization by competing for the binding site

• Protein Stability:

  • Small proteins like FKBP12 (~12 kDa) can be susceptible to degradation

  • Purification buffers may need optimization to maintain stability

  • Inclusion of protease inhibitors and working at lower temperatures is essential

• Homogeneity Requirements for Structural Studies:

  • Structural methods like X-ray crystallography require highly homogeneous samples

  • The dynamic nature of FKBP12, with its flexible loops, may complicate crystallization

  • Size-exclusion chromatography as a final purification step is crucial for achieving monodisperse samples

Strategy for Success:

• Design fusion constructs (e.g., His-MBP-FKBP12) to enhance solubility and facilitate purification

• Express in both prokaryotic and eukaryotic systems to compare yield and quality

• Optimize buffer conditions using thermal shift assays to enhance stability

• Consider including FK506 or analogs during purification to stabilize the protein

• Employ multi-step purification including affinity, ion exchange, and size exclusion chromatography

These approaches address the specific challenges of working with A. gossypii FKBP12 and increase the likelihood of obtaining sufficient quantities of high-quality protein for structural studies.

How might advanced techniques like AlphaFold be used to predict A. gossypii FKBP12-FK506-calcineurin interactions in the absence of crystal structures?

In the absence of crystal structures, AlphaFold and other computational approaches offer powerful alternatives for predicting A. gossypii FKBP12-FK506-calcineurin interactions:

AlphaFold2 Implementation Strategy:

• Individual Protein Structure Prediction:

  • Generate high-confidence models of A. gossypii FKBP12, calcineurin A, and calcineurin B subunits

  • Validate models against known structures of homologous proteins from A. fumigatus (PDB: 6TZ7) and other fungi

  • Assess model quality using metrics like pLDDT scores and predicted aligned error

• Protein-Protein Complex Prediction:

  • Use AlphaFold-Multimer or RoseTTAFold-Complex to model the FKBP12-calcineurin interaction

  • Incorporate evolutionary constraints from multiple sequence alignments of fungal proteins

  • Compare predicted interfaces with known fungal FKBP12-calcineurin complexes

• Ligand Docking Integration:

  • Employ molecular docking software (Autodock Vina, GOLD, Glide) to model FK506 binding

  • Use hybrid approaches combining AlphaFold predictions with traditional docking

  • Validate docking poses against known FK506-FKBP12 structures

Complementary Computational Approaches:

• Molecular Dynamics Simulations:

  • Perform long-timescale simulations of predicted complexes to assess stability

  • Analyze binding free energies using methods like MM-PBSA or FEP

  • Investigate dynamics of key loops (40s, 50s, 80s) that influence binding

• Machine Learning-Based Interface Prediction:

  • Train machine learning models on known FKBP12-calcineurin interfaces

  • Apply models to predict A. gossypii-specific interaction features

  • Integrate predictions with AlphaFold structural models

Experimental Validation Plan:

Applications of Predicted Models:

• Virtual screening for A. gossypii-specific inhibitors targeting the unique aspects of its FKBP12-calcineurin interface

• Rational design of mutations to test structural hypotheses about species-specific interactions

• Guiding experimental structure determination by providing molecular replacement models for X-ray crystallography

This integrative computational approach offers valuable structural insights while experimental structures are being pursued, accelerating the development of A. gossypii-specific inhibitors.

What are the potential applications of A. gossypii FKBP12 research in developing novel antifungal strategies?

Research on A. gossypii FKBP12 has several promising applications for developing novel antifungal strategies:

Selective Antifungal Development:

• Non-Immunosuppressive FK506 Derivatives:

  • Design analogs that exploit structural differences between fungal and human FKBP12

  • Focus on modifications that interact with F88 in fungal FKBP12 rather than H88 in human FKBP12

  • L685,818 provides proof-of-concept that such selective inhibitors are feasible

• Dimerization Disruptors:

  • Target the unique dimerization interface observed in fungal FKBP12 proteins

  • Design small molecules that specifically bind to the 80s loop

  • Since dimerization is not observed in human FKBP12, this approach offers inherent selectivity

• Fungal-Specific Calcineurin Binding:

  • Develop compounds that specifically interfere with the fungal FKBP12-calcineurin interface

  • Focus on interface residues that differ between fungal and human complexes

Biotechnological Applications:

• Engineered Protein Production:

  • Utilize A. gossypii as a platform for producing recombinant proteins

  • Knowledge of FPR1/FKBP12 function could improve protein folding and secretion

  • A. gossypii has already been explored as a host for recombinant protein production

• Conditional Expression Systems:

  • Develop FK506-responsive gene expression systems for biotechnological applications

  • Use engineered FKBP12 variants as molecular switches for controlling gene expression

  • Such systems could be valuable for controlled production of industrial enzymes

Cross-Species Applications:

• Broad-Spectrum Antifungals:

  • Identify conserved features of FKBP12 across multiple fungal pathogens

  • Target these conserved elements to develop broad-spectrum antifungals

  • Address the growing problem of invasive fungal infections in immunocompromised patients

• Species-Selective Targeting:

  • Exploit species-specific variations in FKBP12 structure

  • For example, the Y88 in Mucor circinelloides FKBP12 versus F88 in other fungi

  • Develop compounds tailored to specific fungal pathogens based on their unique FKBP12 features

• Combination Therapies:

  • Design strategies that combine FKBP12 inhibitors with other antifungal agents

  • Target multiple cellular pathways simultaneously to reduce resistance development

  • Exploit synergistic effects between calcineurin inhibition and other antifungal mechanisms

These applications demonstrate the potential of A. gossypii FKBP12 research to contribute to both antifungal drug development and biotechnological innovation, addressing important clinical and industrial challenges.