Recombinant Dictyostelium discoideum Fido domain-containing protein DDB_G0283145 (DDB_G0283145)

What is the Fido domain-containing protein DDB_G0283145?

Fido domain-containing protein DDB_G0283145 is a 143-amino acid protein from the social amoeba Dictyostelium discoideum that belongs to the Fido (FIC domain) protein family. It contains the characteristic AMPylation domain common to fic, doc, and AvrB proteins . This protein family was previously misannotated as laminin A in databases (including pfam04916.1), but has since been recognized as a novel family with AMPylation activity . The protein contains a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to its AMPylation function, a post-translational modification where AMP is covalently attached to target proteins .

How is the Fido domain evolutionarily conserved?

The Fido domain represents an evolutionarily ancient protein fold that appears in diverse species. Phylogenetic analysis reveals:

• Fido domains are found in bacteria, some archaea, and metazoan eukaryotes

• Homologous genes are present in mammals (humans, mice), insects (Drosophila), nematodes (C. elegans), and protozoans (Giardia)

• Interestingly, these domains are absent in yeast, suggesting lineage-specific loss

• The domain shares evolutionary ancestry with doc toxins and type III effector AvrB

This conservation pattern suggests that the Fido domain emerged early in evolution and has been maintained across diverse lineages, likely due to its fundamental biochemical function in AMPylation reactions.

Is DDB_G0283145 related to phospholipase activity in Dictyostelium?

While DDB_G0283145 is classified as a Fido domain-containing protein, it appears to be distinct from the phospholipase B (PLB) activity identified in Dictyostelium. The PLB characterized from Dictyostelium is a 65 kDa protein (with a 48 kDa fragment) that removes both fatty-acid chains from phosphatidylcholine . Although both are present in Dictyostelium, the molecular weight (65 kDa vs. 143 aa/~16 kDa) and functional differences suggest they are separate proteins. The PLB represents a novel family distinct from traditional phospholipases, with conserved sequences such as -NSGTYN(S/N)Q- that are not present in DDB_G0283145 .

What is the significance of the HPFxDGNGR motif in DDB_G0283145?

The HPFxDGNGR motif (specifically HPFIDGNGR in DDB_G0283145) is the defining sequence feature of Fido domains and plays a critical role in catalytic function. Research indicates:

• This motif forms part of the active site for AMPylation reactions

• It contributes to binding of ATP substrate during the transfer of AMP to target proteins

• Mutations in this conserved region typically abolish enzymatic activity

• The motif is present in both bacterial and eukaryotic Fido domains, highlighting its functional importance

When designing experiments to study DDB_G0283145 function, site-directed mutagenesis of residues within this motif represents a powerful approach to confirm and characterize its enzymatic activity.

How should recombinant DDB_G0283145 be properly stored and handled?

Optimal storage and handling of recombinant DDB_G0283145 requires specific conditions to maintain protein stability and activity:

• Storage temperature: Store at -20°C/-80°C upon receipt

• Aliquoting: Divide into small working aliquots to avoid repeated freeze-thaw cycles

• Working storage: For short-term use, working aliquots can be stored at 4°C for up to one week

• Buffer composition: The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• Reconstitution: Centrifuge vial briefly before opening, then reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Cryoprotection: Add glycerol to 5-50% final concentration before long-term storage (50% is recommended)

Following these guidelines will help preserve enzymatic activity and prevent degradation during experimental timelines.

What methods can be used to assay AMPylation activity of DDB_G0283145?

Several complementary approaches can be employed to detect and quantify the AMPylation activity of DDB_G0283145:

In vitro AMPylation assay:

• Incubate purified DDB_G0283145 with potential substrate proteins (such as Rho GTPases) in the presence of ATP

• Detect AMP transfer using:

  • ³²P-α-ATP and autoradiography to visualize labeled substrates

  • Anti-AMP antibodies for western blot detection

  • Mass spectrometry to identify modification sites on target proteins

Functional consequence assessment:

• Monitor downstream signaling events following AMPylation of target proteins

• Assess changes in GTPase activity of modified substrates

• Evaluate binding interactions between modified substrates and their partners

Comparative analysis:

• Include positive controls such as VopS, a well-characterized bacterial Fic protein

• Use site-directed mutants of the HPFxDGNGR motif as negative controls

• Compare activity across different potential substrate proteins

These methodological approaches provide rigorous assessment of AMPylation activity and specificity.

How can I optimize expression of active recombinant DDB_G0283145?

Optimizing expression of functionally active DDB_G0283145 requires careful consideration of several factors:

Expression system selection:

• E. coli BL21(DE3) is commonly used for Fido domain proteins

• Consider codon optimization for the expression host

• Alternative eukaryotic expression systems (insect cells, mammalian cells) may provide native folding environment

Induction conditions:

• Test various IPTG concentrations (0.1-1.0 mM)

• Optimize induction temperature (16-37°C)

• Evaluate different induction durations (4-24 hours)

Solubility enhancement:

• Use fusion tags (His, GST, MBP) to improve solubility

• Consider expressing as a fusion with thioredoxin or SUMO

• Test various lysis buffers with different salt concentrations, pH values, and detergents

Purification strategy:

• Initial capture using immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Secondary purification using ion exchange chromatography

• Final polishing with size exclusion chromatography

Systematic optimization of these parameters will help maximize yield of correctly folded, active protein for downstream applications.

What are effective approaches for identifying DDB_G0283145 substrates?

Identifying the physiological substrates of DDB_G0283145 requires a multifaceted approach:

Unbiased proteomic screening:

• Perform in vitro AMPylation reactions using cell lysates and recombinant DDB_G0283145

• Utilize click chemistry approaches with azido-ATP analogs for enrichment of modified proteins

• Identify AMP-modified proteins by mass spectrometry

Candidate-based approaches:

• Test GTPases as potential substrates, based on the known activity of related Fido proteins

• Examine cytoskeletal proteins involved in Dictyostelium motility and development

• Investigate proteins in pathways regulated during Dictyostelium's life cycle

Binding partner identification:

• Affinity purification using DDB_G0283145 as bait

• Yeast two-hybrid screening

• Proximity labeling techniques such as BioID or APEX

Validation experiments:

• Site-directed mutagenesis of putative modification sites

• Functional assays to assess the consequences of AMPylation

• Generation of substrate-specific antibodies that recognize AMP-modified epitopes

These approaches provide complementary evidence for substrate identification and validation.

How can I determine if DDB_G0283145 has phospholipase activity?

To assess whether DDB_G0283145 possesses phospholipase activity (distinct from its predicted AMPylation function), implement the following experimental strategy:

Radio-labeled phospholipid assay:

• Incubate purified DDB_G0283145 with radio-labeled phospholipids (³²P or ¹⁴C-labeled)

• Extract and separate reaction products using thin-layer chromatography

• Quantify liberated fatty acids or water-soluble head groups

Fluorescent substrate assay:

• Use fluorescently labeled phospholipids with quenched fluorescence

• Monitor fluorescence increase upon phospholipid hydrolysis

• Compare kinetics with known phospholipases

Substrate specificity profiling:

• Test activity against various phospholipids (PC, PI, PE)

• Determine positional specificity (PLA₁, PLA₂, PLB)

• Evaluate lysophospholipase activity

Comparative analysis with known PLB:

• Include the 65 kDa Dictyostelium PLB as positive control

• Test inhibitor sensitivity profiles

• Compare pH and cation requirements

These approaches will definitively determine whether DDB_G0283145 possesses phospholipase activity in addition to its predicted AMPylation function.

How does DDB_G0283145 compare structurally with other Fido domain proteins?

Structural analysis of DDB_G0283145 in comparison with other Fido domain proteins reveals important insights into evolutionary relationships and mechanisms:

Conserved structural elements:

Key structural differences:

• DDB_G0283145 (143 aa) is considerably smaller than bacterial Fic proteins (~200-500 aa)

• The protein likely lacks regulatory domains present in some bacterial Fido proteins

• Structural modeling suggests potential differences in substrate binding surfaces

Active site architecture:

These structural comparisons provide insight into the evolutionary adaptations of Fido domains across different organisms and functional contexts .

What is the predicted role of DDB_G0283145 in Dictyostelium discoideum biology?

Based on knowledge of Fido domain functions and Dictyostelium biology, several hypotheses can be formulated about the physiological role of DDB_G0283145:

Development regulation:

• Dictyostelium undergoes a complex developmental cycle involving cell aggregation and differentiation

• DDB_G0283145 may regulate GTPases involved in chemotaxis during aggregation

• AMPylation could serve as a reversible switch in developmental signaling pathways

Cytoskeletal regulation:

• Fido proteins in other organisms modify GTPases controlling actin dynamics

• DDB_G0283145 may regulate Dictyostelium motility and phagocytosis

• The protein could participate in remodeling cell shape during development

Stress response:

• AMPylation often functions in stress response pathways

• DDB_G0283145 might be activated during nutrient limitation

• It could regulate metabolism during the transition from unicellular to multicellular phases

Host-pathogen interactions:

• Dictyostelium serves as a model for phagocytosis and bacterial interactions

• DDB_G0283145 might protect against bacterial effectors

• Alternatively, it could regulate endocytic trafficking during bacterial engulfment

Testing these hypotheses requires techniques like gene knockout, phenotypic analysis, and localization studies under various developmental and stress conditions.

What are the challenges in resolving potential dual enzymatic activities of DDB_G0283145?

Investigating whether DDB_G0283145 possesses both AMPylation and phospholipase activities presents specific methodological challenges:

Activity segregation:

• Design mutants that selectively disrupt one activity while preserving the other

• Perform active site mapping using chemical modification approaches

• Employ partial proteolysis to identify functional domains

Substrate competition assays:

• Test whether phospholipids inhibit AMPylation activity and vice versa

• Analyze reaction kinetics with mixed substrates

• Determine whether both activities share a common catalytic mechanism

Structural studies:

• Crystallize DDB_G0283145 with different substrates or substrate analogs

• Perform molecular docking studies with both nucleotides and phospholipids

• Use HDX-MS (hydrogen-deuterium exchange mass spectrometry) to map substrate binding regions

Controls and benchmarking:

• Compare activities with well-characterized single-function enzymes

• Ensure assay conditions are compatible for detecting both activities

• Validate findings using orthogonal detection methods

Resolving these challenges will provide mechanistic insight into the potential multifunctionality of DDB_G0283145 and related proteins.

How might post-translational modifications affect DDB_G0283145 function?

Post-translational modifications (PTMs) likely play critical roles in regulating DDB_G0283145 activity, localization, and interactions:

Predicted regulatory PTMs:

Experimental approaches:

• Treat recombinant protein with various phosphatases before activity assays

• Generate phosphomimetic mutations at predicted sites

• Compare activity of full-length protein with naturally occurring truncated forms

• Identify PTMs using mass spectrometry under different cellular conditions

Regulatory implications:

• PTMs may allow switching between different enzymatic activities

• Modifications could direct subcellular localization

• PTMs might respond to developmental signals or stress conditions

Understanding the PTM landscape will provide critical insight into the contextual regulation of DDB_G0283145 function during Dictyostelium's life cycle.

What approaches can determine the subcellular localization and dynamics of DDB_G0283145?

Determining the subcellular localization and dynamics of DDB_G0283145 requires multiple complementary approaches:

Imaging techniques:

• Generate fluorescent protein fusions (GFP, mCherry) for live-cell imaging

• Perform immunofluorescence using antibodies against the native protein or epitope tags

• Use super-resolution microscopy to resolve detailed localization patterns

• Implement FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

Biochemical fractionation:

• Perform differential centrifugation to separate cellular compartments

• Use density gradient fractionation for membrane compartment separation

• Isolate specific organelles using magnetic immunoprecipitation

• Analyze fractions by western blotting for DDB_G0283145

Signal sequence analysis:

• The protein contains a predicted signal sequence with cleavage site between positions 20-21 (VLS–QS)

• Generate signal sequence mutants to test localization requirements

• Use reporter constructs to validate signal sequence functionality

• Compare with predicted transmembrane domains or localization signals

Dynamic regulation studies:

• Track localization changes during Dictyostelium development

• Monitor responses to cellular stresses (nutrient limitation, osmotic stress)

• Assess colocalization with potential substrates under various conditions

These approaches will reveal not only where DDB_G0283145 functions, but also how its localization is regulated in response to cellular needs.

Why might recombinant DDB_G0283145 show inconsistent enzymatic activity?

Inconsistent enzymatic activity of recombinant DDB_G0283145 can stem from several factors that researchers should systematically address:

Protein quality issues:

• Improper folding due to rapid expression or inclusion body formation

• Aggregation during purification or storage

• Loss of essential cofactors during purification

• Proteolytic degradation (add protease inhibitors during purification)

Assay condition variables:

• Buffer composition effects (pH, salt concentration, presence of detergents)

• Temperature sensitivity of the enzyme

• Requirement for specific divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

• Substrate quality or concentration issues

Methodological solutions:

• Validate protein folding using circular dichroism spectroscopy

• Assess oligomeric state using size exclusion chromatography

• Test enzyme activity immediately after purification

• Optimize buffer conditions systematically

• Include proper positive controls in each experiment

Storage and handling improvements:

• Store small aliquots to avoid freeze-thaw cycles

• Add stabilizing agents (glycerol, reducing agents)

• Test activity retention over time under different storage conditions

• Consider flash-freezing in liquid nitrogen

Implementing these approaches will help ensure reproducible activity in experimental applications.

How can I distinguish between AMPylation and other post-translational modifications?

Differentiating AMPylation from other post-translational modifications requires specific analytical approaches:

Mass spectrometry-based discrimination:

• AMPylation adds a mass of 329 Da (AMP) to modified residues

• This mass shift is distinct from common PTMs like phosphorylation (80 Da) or acetylation (42 Da)

• MS/MS fragmentation patterns of AMP-modified peptides show characteristic neutral losses

• Use high-resolution MS to resolve AMPylation from similar modifications

Chemical and enzymatic approaches:

• AMPylation is resistant to phosphatases but may be sensitive to specific phosphodiesterases

• Use differential chemical stability tests (pH, hydroxylamine sensitivity)

• Develop AMPylation-specific antibodies for immunological detection

• Employ nucleotide-specific reagents that recognize the adenosine moiety

Control experiments:

• Include catalytically inactive DDB_G0283145 mutants (H65A in the HPF motif)

• Perform reactions with and without ATP

• Compare with known AMPylating enzymes (VopS) and their substrates

• Use modified substrates with mutations at potential modification sites

These approaches provide multiple lines of evidence to confidently identify AMPylation events.

What controls are essential when characterizing DDB_G0283145 function?

Rigorous control experiments are crucial for reliable characterization of DDB_G0283145 function:

Protein quality controls:

• SDS-PAGE and western blot to confirm protein integrity

• Size exclusion chromatography to assess oligomerization state

• Circular dichroism to verify proper folding

• Activity assays with known functional Fido proteins as benchmarks

Enzymatic activity controls:

• Catalytically inactive mutant (H65A in HPF motif)

• Substrate-binding mutants

• Reactions with and without ATP/Mg²⁺

• Heat-inactivated enzyme preparations

Substrate specificity controls:

• Test non-physiological substrates as negative controls

• Include substrates of known Fido proteins as positive controls

• Use modified substrates with mutations at putative target sites

• Compare in vitro vs. in vivo modification patterns

Expression system controls:

• Empty vector controls in expression experiments

• Unrelated proteins purified under identical conditions

• Commercially available standards where applicable

• Multiple independently expressed protein batches to ensure reproducibility

These comprehensive controls ensure that observed phenotypes and activities can be confidently attributed to DDB_G0283145 function.

What are common pitfalls in analyzing protein-protein interactions involving DDB_G0283145?

When investigating protein-protein interactions of DDB_G0283145, researchers should be aware of several common pitfalls:

Technical challenges:

• Non-specific binding to affinity matrices or tags

• Detergent sensitivity of genuine interactions

• Transient or weak interactions may be lost during washing steps

• Buffer conditions may disrupt physiologically relevant interactions

Biological complexities:

• Interactions may be modification-dependent (requiring active AMPylation)

• Binding may be developmental stage-specific

• Subcellular compartmentalization may prevent interactions in lysates

• Additional factors may be required to mediate interactions

Methodological solutions:

• Include multiple negative controls (unrelated proteins, tag-only controls)

• Validate interactions using orthogonal methods (Y2H, co-IP, FRET, SPR)

• Test interactions under various buffer conditions

• Perform crosslinking to capture transient interactions

• Use domain mapping to identify specific interaction regions

Data interpretation considerations:

• Distinguish between direct and indirect interactions

• Consider substrate relationships vs. stable binding partners

• Evaluate stoichiometry of observed interactions

• Assess functional relevance through activity assays

How can I address difficulties in detecting low-abundance or transient AMPylation events?

Detecting low-abundance or transient AMPylation events mediated by DDB_G0283145 requires specialized approaches:

Enrichment strategies:

• Develop AMPylation-specific antibodies for immunoprecipitation

• Use clickable ATP analogs (azido-ATP) for bioorthogonal labeling and enrichment

• Implement substrate trapping with catalytically compromised mutants

• Generate engineered substrates with enhanced binding but reduced turnover

Enhanced detection methods:

• Employ highly sensitive targeted mass spectrometry (PRM or MRM)

• Use AQUA peptides as internal standards for quantification

• Implement SILAC or TMT labeling for comparative proteomics

• Develop fluorescent sensors for real-time AMPylation monitoring

Kinetic analysis approaches:

• Use rapid quench-flow techniques to capture short-lived intermediates

• Perform pulse-chase experiments to track modification dynamics

• Develop computational models to predict modification kinetics

• Implement temperature-jump methods to study reaction mechanisms

Cellular context optimization:

• Use inhibitors of deAMPylases to stabilize modifications

• Synchronize cells to capture stage-specific events

• Apply stressors known to enhance AMPylation activity

• Express substrates at higher levels to facilitate detection

These approaches enhance sensitivity and temporal resolution for detecting physiologically relevant AMPylation events.

What are the most promising approaches for elucidating DDB_G0283145 function in vivo?

Future research to elucidate the in vivo function of DDB_G0283145 should prioritize these approaches:

Genetic manipulation:

• Generate knockout strains using CRISPR-Cas9

• Create conditional expression systems for temporal control

• Develop complementation systems with mutant variants

• Implement tissue-specific or development-stage-specific expression

Phenotypic characterization:

• Analyze growth, development, and multicellular morphogenesis

• Assess stress responses and survival under various conditions

• Evaluate phagocytosis, chemotaxis, and cytoskeletal dynamics

• Measure interactions with bacterial pathogens

Systems biology approaches:

• Perform global proteomics to identify AMPylation targets

• Analyze transcriptome changes in knockout vs. wild-type

• Map the interactome under different developmental conditions

• Integrate with existing Dictyostelium 'omics datasets

Comparative studies:

• Examine functions of homologs in other species

• Perform cross-species complementation experiments

• Compare with other Dictyostelium Fido domain proteins

• Evaluate evolutionary patterns of substrate specificity

These multifaceted approaches will provide comprehensive insight into the biological significance of DDB_G0283145 in Dictyostelium biology and broader evolutionary context.

How might understanding DDB_G0283145 inform therapeutic approaches targeting Fido domain proteins?

Insights from DDB_G0283145 research could inform therapeutic strategies targeting Fido domain proteins in various contexts:

Therapeutic potential:

• Human Fido domain proteins may represent novel drug targets

• Bacterial Fic proteins are virulence factors in several pathogens

• AMPylation processes may be dysregulated in certain diseases

• Understanding conserved mechanisms could enable broad-spectrum approaches

Drug development strategies:

• Use DDB_G0283145 structure as template for homology modeling of human homologs

• Develop high-throughput screens based on AMPylation activity

• Design peptide inhibitors targeting conserved substrate binding regions

• Create small molecule inhibitors of the ATP binding pocket

Translational applications:

• Anti-virulence therapeutics targeting bacterial Fic proteins

• Modulators of human Fido proteins for cellular engineering

• Diagnostic tools based on AMPylation detection

• Research reagents for studying post-translational regulation

While DDB_G0283145 itself may not be a therapeutic target, the fundamental mechanisms revealed through its study could significantly advance our ability to target this protein family in clinical contexts.