Recombinant Drosophila pseudoobscura pseudoobscura Adenosine monophosphate-protein transferase FICD homolog (GA21854)

What is the FICD homolog in Drosophila pseudoobscura and what is its primary function?

The FICD (Filamentation Induced by cAMP Domain) homolog in Drosophila pseudoobscura is an adenosine monophosphate-protein transferase that catalyzes both AMPylation and deAMPylation reactions. AMPylation is a post-translational modification where adenosine monophosphate (AMP) is transferred from adenosine triphosphate (ATP) to target proteins, serving as a critical regulatory mechanism in cellular functions. According to enzyme classification, FICD is classified as an adenylyltransferase enzyme (EC 2.7.7.108) and catalyzes reactions annotated as Rhea 55932 and Rhea 54292 . The protein plays a significant role in the endoplasmic reticulum unfolded protein response pathway, particularly through its interaction with chaperone proteins such as BiP/HSPA5 .

How does the protein structure of FICD in D. pseudoobscura contribute to its function?

The FICD protein in D. pseudoobscura contains a Fido domain (residues 285-420) which mediates its adenylyltransferase activity. Key structural features include numerous hydrogen bonds that maintain protein stability and function. For instance, in wild-type FICD, Asn339 forms three hydrogen bonds (with Val285, Val335, and Gln336), while Ser340 establishes three bonds (with Glu342 and Trp337) . The protein functions as a homodimer, with dimerization partially regulating its dual enzymatic activity - the AMPylation and deAMPylation of target proteins. During homodimerization, FICD forms multiple hydrogen bonds including interactions between Ala252 and Lys256, Leu258 and Arg250, and Asn262 with itself . This dimerization is crucial for modulating the enzyme's deAMPylation activity.

What experimental methods are commonly used to study recombinant FICD in Drosophila systems?

For studying recombinant FICD in Drosophila systems, researchers typically employ a combination of molecular genetic and biochemical techniques. Recombinant protein expression systems can be established using bacterial or insect cell expression platforms. For structural analysis, techniques like X-ray crystallography or, as demonstrated in recent research, computational structural prediction using algorithms such as AlphaFold can be employed to determine protein conformations and predict functional interactions . Functional characterization often involves enzymatic assays measuring AMPylation/deAMPylation activities. Protein-protein interaction studies using techniques such as co-immunoprecipitation or yeast two-hybrid systems help identify binding partners like BiP/HSPA5 . In vivo, researchers may use genetic approaches including transgenesis and gene editing in Drosophila models to assess physiological functions and phenotypic impacts.

How does the dual AMPylation/deAMPylation function of FICD regulate the unfolded protein response in Drosophila pseudoobscura?

The dual enzymatic activity of FICD plays a sophisticated role in regulating the unfolded protein response (UPR) in Drosophila pseudoobscura, primarily through its interaction with BiP/HSPA5 chaperone proteins. As evidenced by protein-protein interaction analyses, FICD forms specific hydrogen bonds with BiP, modifying its activity state through reversible AMPylation . During normal cellular conditions, FICD AMPylates BiP, reducing its chaperone activity and maintaining ER homeostasis. Under ER stress conditions, FICD shifts toward its deAMPylation function, removing AMP from BiP and thereby activating it to address unfolded protein accumulation.

This regulatory mechanism is integrated into multiple cellular pathways, including IRE1-mediated unfolded protein response (GO:0036498), regulation of endoplasmic reticulum unfolded protein response (GO:1900101), and cellular response to topologically incorrect protein (GO:0035967) . The dimerization state of FICD is critical for balancing these dual activities, with research showing that loss of dimerization leads to increased AMPylation and reduced deAMPylation of BiP. This molecular switch mechanism allows for rapid, reversible response to changing ER stress conditions, positioning FICD as a key regulator in proteostasis maintenance rather than merely an enzyme with two distinct catalytic capabilities.

How can site-directed mutagenesis be used to investigate the functional domains of the FICD homolog in D. pseudoobscura?

Site-directed mutagenesis provides a powerful approach for dissecting the functional architecture of D. pseudoobscura FICD. Based on structural insights, researchers should strategically target several key regions of the protein:

The Fido domain (residues 285-420) can be systematically mutated to disrupt the adenylyltransferase activity. Specifically, researchers should focus on residues like Asn339 and Ser340, which form multiple hydrogen bonds crucial for structural integrity . By altering these residues, one can evaluate their contribution to catalytic function and substrate specificity.

The dimerization interface should be targeted by mutating residues such as Ala252, Lys256, Leu258, and Asn262, which form hydrogen bonds during homodimerization . Since dimerization regulates the balance between AMPylation and deAMPylation activities, mutations disrupting this interface would help elucidate how quaternary structure influences enzymatic function.

For investigating BiP interaction, mutations should target residues involved in FICD-BiP binding, such as Thr80, Ser83, and Asn111 . The experimental design should include:

• Generation of mutant constructs using PCR-based site-directed mutagenesis

• Expression of wild-type and mutant proteins in a suitable system

• Comparative analysis of enzymatic activities (AMPylation/deAMPylation)

• Protein-protein interaction assays to assess binding with BiP

• Structural analysis using techniques like circular dichroism to detect conformational changes

This approach would reveal structure-function relationships and provide mechanistic insights into how FICD regulates the unfolded protein response.

What are the optimal conditions for expressing and purifying recombinant D. pseudoobscura FICD homolog for structural studies?

For expressing and purifying recombinant D. pseudoobscura FICD homolog with optimal yield and activity, researchers should implement a systematic approach tailored to this protein's characteristics. Based on the protein's structural features, including its homodimerization tendency and multiple hydrogen bonding networks , the following protocol is recommended:

Expression System Selection:

• Bacterial systems using E. coli BL21(DE3) strains are suitable for initial expression trials

• For complex folding requirements, consider insect cell expression systems (Sf9 or Hi5 cells) which better approximate the native folding environment of Drosophila proteins

Expression Optimization:

• Test multiple fusion tags (His6, GST, MBP) with proteolytic cleavage sites

• Evaluate expression at lower temperatures (16-20°C) to enhance proper folding

• Consider co-expression with chaperones if misfolding occurs

• Use auto-induction media for bacterial expression to achieve higher cell density

Purification Strategy:

• Initial capture using affinity chromatography based on the fusion tag

• Intermediate purification using ion exchange chromatography (consider the theoretical pI of FICD)

• Polishing step using size exclusion chromatography to separate monomeric and dimeric forms

• Include reducing agents (1-5 mM DTT or TCEP) throughout purification to prevent oxidation

• Maintain pH range of 7.0-8.0 based on the optimal stability range for similar proteins

Stability Enhancement:

• Screen buffer conditions using differential scanning fluorimetry

• Test stabilizing additives including glycerol (10-20%), low concentrations of detergents, and specific metal ions

• For structural studies, evaluate protein stability in the presence of substrate analogs or inhibitors

This methodological approach should yield protein suitable for structural studies including X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy.

How can electrophoretic mobility analysis be applied to study D. pseudoobscura FICD variants?

Electrophoretic mobility analysis offers a valuable approach for characterizing D. pseudoobscura FICD variants, leveraging principles established in previous Drosophila protein studies. Research has demonstrated that the average effect of an amino acid charge change on mobility of proteins in D. pseudoobscura is approximately 0.046 units . This quantitative relationship can be exploited to analyze FICD variants with the following methodology:

First, researchers should employ Ferguson plots to separate charge and shape components of mobility differences, as this technique effectively distinguishes between mobility changes caused by charge alterations versus conformational changes . For FICD variants, this is particularly relevant since mutations may affect both the protein's charge and its homodimerization properties.

A systematic approach would involve:

• Generating FICD variants with targeted mutations, particularly in the Fido domain (residues 285-420)

• Performing electrophoresis under varying gel concentrations to construct Ferguson plots

• Calculating relative mobility values and correlating them with predicted charge changes

• Using the established charge-mobility relationship (0.046 units per charge) to validate structural predictions

This methodology could be particularly valuable for investigating how mutations affect FICD's functional states, especially since the protein's dimerization is crucial for regulating its dual AMPylation/deAMPylation activities . By comparing experimental mobility data with predictions from computational models like AlphaFold, researchers can validate structural hypotheses about how specific mutations impact FICD function.

What evolutionary insights can be gained from comparing the FICD homolog across different Drosophila species?

Evolutionary analysis of FICD across Drosophila species provides valuable insights into both conservation of critical functional domains and adaptive evolution of regulatory mechanisms. D. pseudoobscura represents a particularly interesting model for such studies due to its distinctive genomic characteristics. The species exhibits significantly higher recombination rates compared to D. melanogaster and even its close relative D. persimilis, with rates sometimes reaching 50 kb/cM . This elevated recombination rate potentially allows for more efficient selection and faster adaptation of functional protein domains.

Comparative analysis should focus on:

• Conservation of the Fido domain (residues 285-420) across species, as this region mediates adenylyltransferase activity

• Variation in dimerization interfaces, particularly residues involved in hydrogen bonding during homodimerization (Ala252, Lys256, Leu258, and Asn262)

• Evolution of BiP-binding regions, which are critical for FICD's role in regulating the unfolded protein response

The high recombination rates in D. pseudoobscura potentially reduce the impact of interference selection, allowing more rapid evolution of functionally important domains . This could lead to lineage-specific adaptations in the FICD homolog, particularly in regions that modulate the balance between AMPylation and deAMPylation activities. Integrating this evolutionary analysis with structural insights can reveal how natural selection has shaped this protein's dual functionality across Drosophila lineages.

How does the interaction between FICD and BiP influence experimental design for studying the unfolded protein response in Drosophila pseudoobscura?

The well-established interaction between FICD and BiP (HSPA5) represents a central axis for designing experiments to study the unfolded protein response (UPR) in Drosophila pseudoobscura. Structural predictions have revealed specific hydrogen bond interactions between these proteins, including those involving Thr80, Ser83, and Asn111 of FICD with corresponding residues in BiP . These molecular interactions directly influence experimental approaches in several ways.

When designing experiments to investigate the UPR in D. pseudoobscura, researchers should:

• Implement co-immunoprecipitation studies optimized for preserving the FICD-BiP interaction, using crosslinking approaches that capture transient interactions during AMPylation/deAMPylation cycles

• Develop fluorescent biosensors that can monitor the AMPylation state of BiP in real-time, allowing dynamic visualization of FICD activity during ER stress responses

• Establish genetic models with mutations specifically targeting the FICD-BiP interface rather than catalytic domains alone, as these may preserve enzymatic activity while disrupting the physiologically relevant protein-protein interactions

• Design cell-based assays that simultaneously monitor key UPR pathways, including IRE1-mediated responses (GO:0036498) and cellular responses to topologically incorrect proteins (GO:0035967)

• Incorporate quantitative mass spectrometry approaches to detect changes in the AMPylation profile of BiP under various stress conditions

This experimental framework acknowledges that FICD's role in the UPR extends beyond its enzymatic activities to include specific protein interaction networks that modulate cellular stress responses. By designing experiments that capture both the enzymatic and scaffolding functions of FICD, researchers can develop a more comprehensive understanding of how this protein coordinates the UPR in Drosophila pseudoobscura.

What are the current limitations in studying the function of FICD homolog in D. pseudoobscura and how might they be addressed?

Current research on the FICD homolog in D. pseudoobscura faces several technical and conceptual limitations that require innovative approaches to overcome. A primary challenge is the difficulty in distinguishing between the protein's AMPylation and deAMPylation activities in vivo, as these opposing functions can mask each other in experimental settings. Furthermore, the high recombination rates in D. pseudoobscura (sometimes reaching 50 kb/cM) complicate genetic approaches by potentially introducing rapid changes in experimental populations.

To address these limitations, researchers should consider implementing:

• Development of activity-specific antibodies that selectively recognize the AMPylating versus deAMPylating conformational states of FICD

• Application of CRISPR-Cas9 genome editing to generate separation-of-function mutations that selectively disrupt either AMPylation or deAMPylation activities

• Design of substrate-trapping mutants that can capture transient FICD-target interactions during the catalytic cycle

• Implementation of single-cell approaches to account for cell-to-cell variability in FICD function during stress responses

• Integration of computational modeling with experimental validation to predict how specific mutations affect the balance between AMPylation and deAMPylation

Additionally, functional studies in multiple genetic backgrounds are essential, as demonstrated by research showing that structural variations in FICD can affect both homodimerization and interaction with BiP, potentially disrupting critical cellular pathways . Addressing these limitations will require multidisciplinary approaches that combine structural biology, biochemistry, and advanced genetic techniques.

How can protein structure prediction tools like AlphaFold be effectively integrated into experimental research on D. pseudoobscura FICD?

Integrating protein structure prediction tools like AlphaFold with experimental research on D. pseudoobscura FICD creates powerful synergies for advancing our understanding of this protein's function. The successful application of AlphaFold in predicting structural changes in FICD variants demonstrates the value of this approach . To maximize the utility of these computational tools, researchers should implement a systematic framework:

This approach has already shown promise, as demonstrated in recent research where AlphaFold successfully predicted structural differences in FICD protein variants, including changes in hydrogen bonding patterns that may affect function . The quantitative nature of these predictions allows researchers to prioritize experimental efforts, focusing on mutations most likely to yield mechanistic insights. This computational-experimental integration represents a particularly valuable approach for studying proteins like FICD where subtle structural changes can have significant functional consequences.

What is the relationship between recombination rates in D. pseudoobscura and genetic studies of FICD function?

The exceptionally high recombination rates in Drosophila pseudoobscura present both opportunities and challenges for genetic studies of FICD function. With recombination rates significantly higher than in D. melanogaster and even D. persimilis, sometimes reaching 50 kb/cM , D. pseudoobscura offers unique advantages for fine-scale genetic mapping studies of FICD and its interacting partners.

This high recombination environment affects FICD research in several critical ways:

• Enhanced Mapping Resolution:
  The high recombination rate allows for extremely fine-scale genetic mapping, enabling researchers to precisely locate regulatory elements and functional domains affecting FICD expression and activity. This is particularly valuable when studying subtle phenotypic effects of FICD variants.

• Reduced Linkage Disequilibrium:
  The elevated recombination rate reduces linkage disequilibrium around the FICD locus, allowing more precise association of specific genetic variants with functional outcomes. This provides cleaner genetic backgrounds for studying specific FICD variants.

• Experimental Design Considerations:
  When designing genetic crosses for FICD studies, researchers should account for the high recombination rates by:

  • Using higher marker densities around the FICD locus

  • Employing smaller mapping intervals for fine-structure analysis

  • Considering the increased potential for recombination between closely linked markers

• Evolutionary Context:
  The high recombination environment may have influenced the evolutionary trajectory of FICD in D. pseudoobscura compared to other Drosophila species, potentially affecting its functional specialization .

These considerations highlight how understanding the genomic context of D. pseudoobscura, particularly its recombinational landscape, is essential for designing effective genetic studies of FICD function and for interpreting results in an appropriate evolutionary context.

What controls should be included when studying the enzymatic activity of recombinant D. pseudoobscura FICD?

When studying the enzymatic activity of recombinant D. pseudoobscura FICD, a comprehensive control strategy is essential to ensure reliable and interpretable results. Based on FICD's dual AMPylation and deAMPylation activities and structural characteristics , the following controls should be systematically implemented:

Enzymatic Activity Controls:

• Catalytic-dead mutants: Include H363A mutations (or equivalent in D. pseudoobscura FICD) which abolish catalytic activity as negative controls

• Selective activity mutants: Use E234G mutations (or equivalent) which enhance AMPylation and reduce deAMPylation to distinguish between the two activities

• Substrate controls: Include both BiP and non-physiological substrates to assess specificity

• Time-course analysis: Measure activity at multiple time points to distinguish initial rates from equilibrium states

Structural Integrity Controls:

• Dimerization controls: Since FICD functions as a homodimer , include both wild-type dimers and forced monomers through interface mutations

• Thermal stability assays: Conduct differential scanning fluorimetry to confirm proper folding of recombinant proteins

• Size exclusion chromatography: Verify oligomeric state of purified proteins

Experimental Design Controls:

• Buffer composition variations: Test activity across different pH, salt, and divalent cation concentrations

• Temperature dependence: Assess activity at both physiological (25°C for Drosophila) and standard laboratory temperatures

• ATP analogs: Use non-hydrolyzable ATP analogs as competitive inhibitors to confirm ATP-dependent mechanisms

This comprehensive control strategy addresses both technical considerations in working with recombinant proteins and the specific biological complexities of FICD's dual enzymatic activities. By systematically implementing these controls, researchers can confidently differentiate genuine enzymatic activities from artifacts and establish a foundation for detailed mechanistic studies.

How can researchers effectively compare FICD function between D. pseudoobscura and model organisms like D. melanogaster?

To effectively compare FICD function between D. pseudoobscura and model organisms like D. melanogaster, researchers should implement a multi-level comparative approach that accounts for genomic, structural, and functional differences. This systematic comparison should include:

Genomic Context Analysis:

• Assess synteny and gene structure conservation between species

• Compare recombination rates around the FICD locus, considering that D. pseudoobscura has significantly higher recombination rates than D. melanogaster

• Examine regulatory element conservation to identify potentially divergent expression patterns

Protein Structure-Function Comparison:

• Generate recombinant proteins from both species and compare:

  • Enzymatic kinetics for both AMPylation and deAMPylation activities

  • Substrate specificity profiles

  • Dimerization properties and stability

• Use comparative structural models to identify species-specific differences in key functional domains

• Perform complementation experiments by expressing each species' FICD in the heterologous background

In Vivo Functional Analysis:

• Develop equivalent genetic tools in both species:

  • CRISPR-engineered mutants with identical mutations

  • Fluorescently-tagged FICD for localization studies

  • Reporter systems for UPR activation

• Compare phenotypic outcomes of FICD disruption, particularly in tissues with high secretory demands

• Assess stress response dynamics using standardized ER stress induction protocols

This comparative approach leverages the experimental advantages of each species: the genetic tractability and extensive toolkit of D. melanogaster, and the unique genomic characteristics of D. pseudoobscura with its high recombination rates . By systematically comparing FICD function across these species, researchers can identify both conserved mechanisms and lineage-specific adaptations in this important regulatory enzyme.

What data analysis approaches are recommended for interpreting results from FICD mutation studies in D. pseudoobscura?

For interpreting results from FICD mutation studies in D. pseudoobscura, researchers should implement a multi-layered data analysis framework that integrates structural predictions with functional outcomes. Based on the complex dual functionality of FICD and its crucial role in protein homeostasis , the following analytical approaches are recommended:

Structure-Function Correlation Analysis:

• Develop quantitative scoring systems that correlate predicted structural changes (using tools like AlphaFold) with measured functional outcomes

• Implement computational pipelines that analyze hydrogen bonding networks in wild-type and mutant proteins

• Cluster mutations based on their effects on dimerization, BiP interaction, and catalytic activity

Statistical Approaches for Phenotypic Data:

• Apply hierarchical mixed-effects models that account for genetic background variation

• Implement time-series analysis for stress response experiments

• Use multivariate analysis to simultaneously evaluate multiple UPR parameters

• Correct for multiple testing when screening numerous mutations

Visualization Strategies:

• Generate structure-function heat maps overlaying mutation positions with their functional impact

• Develop interaction network visualizations showing how FICD mutations affect broader cellular pathways

• Create comparative visualization tools for cross-species functional conservation

Integrative Data Analysis:

• Implement machine learning approaches to identify patterns in complex phenotypic data

• Integrate proteomic and transcriptomic data to assess global impacts of FICD mutations

• Develop pathway enrichment analyses specific to ER stress responses

This comprehensive analytical framework enables researchers to extract maximum insight from FICD mutation studies, moving beyond simple binary assessments of function/dysfunction toward a mechanistic understanding of how specific structural alterations affect FICD's dual enzymatic activities and its role in cellular stress responses.

What structural features of FICD are critical for experimental design?

Understanding the key structural features of FICD is essential for designing effective experiments. Based on structural analyses, the following table summarizes critical domains and interactions that should guide experimental approaches:

Table 1: Key Structural Features of FICD for Experimental Consideration

This structural information provides a roadmap for experimental design, highlighting regions where targeted mutations can provide insight into FICD function. For instance, mutations in the dimerization interface could help elucidate how quaternary structure influences the balance between AMPylation and deAMPylation activities, while modifications to BiP interaction sites could reveal mechanisms of substrate recognition and specificity.

How do hydrogen bonding patterns in wild-type versus mutant FICD affect experimental outcomes?

Hydrogen bonding patterns in FICD play a critical role in determining protein structure, stability, and function, with significant implications for experimental outcomes. Comparative analysis of wild-type and mutant FICD variants reveals specific hydrogen bonding differences that researchers should consider when designing and interpreting experiments:

Table 2: Comparative Hydrogen Bonding Patterns in Wild-Type vs. Mutant FICD

What are the comparative recombination rates across Drosophila species and how do they impact FICD studies?

Recombination rates vary significantly across Drosophila species, with important implications for genetic studies of FICD. The following table presents comparative recombination data that researchers should consider when designing experiments:

Table 3: Comparative Recombination Rates in Drosophila Species

These differential recombination rates have significant implications for FICD studies:

• The high recombination rate in D. pseudoobscura (sometimes reaching 50 kb/cM) provides exceptional mapping resolution, making it ideal for fine-structure genetic analysis of FICD function

• When designing mapping experiments, researchers can achieve higher resolution with smaller sample sizes in D. pseudoobscura compared to other Drosophila species

• The elevated recombination rate reduces linkage disequilibrium, allowing more precise association between genetic variants and phenotypic outcomes

• For comparative studies, researchers must account for these recombination differences when designing equivalent mapping approaches across species

• Interspecies hybrids offer particularly high recombination rates in collinear regions, potentially providing even greater mapping resolution for specific experimental questions

Understanding these species-specific recombination landscapes is essential for optimizing genetic approaches to FICD functional studies and for correctly interpreting mapping data in evolutionary contexts.

What are the most promising future research directions for studying FICD in Drosophila pseudoobscura?

Based on current knowledge and technological capabilities, several promising research directions emerge for advancing our understanding of FICD in Drosophila pseudoobscura. The unique characteristics of D. pseudoobscura, including its exceptionally high recombination rates , combined with emerging insights into FICD's dual enzymatic functions , create opportunities for innovative research approaches.

Priority research directions should include developing comprehensive models of how FICD's AMPylation/deAMPylation balance regulates the unfolded protein response across different tissues and developmental stages. This will require integrating structural insights from computational models with in vivo functional studies. The high recombination rate in D. pseudoobscura offers exceptional resolution for mapping genetic modifiers of FICD function, potentially revealing new components of this regulatory network .

Comparative evolutionary studies across Drosophila species may reveal how FICD function has adapted to different ecological niches, particularly in stress response pathways. Furthermore, exploring the interaction network beyond the established FICD-BiP axis could uncover additional substrates and regulatory partners. Finally, leveraging the genetic tractability of Drosophila to model human FICD-related pathologies represents an important translational direction, as mutations in this conserved pathway have been implicated in neurological and developmental disorders .