Recombinant Drosophila willistoni Ubiquitin-like modifier-activating enzyme 5 (GK10218)

What is Ubiquitin-like modifier-activating enzyme 5 (GK10218) in Drosophila willistoni and how does it function molecularly?

Ubiquitin-like modifier-activating enzyme 5 (GK10218) in Drosophila willistoni serves as an E1 enzyme in the UFMylation pathway. It catalyzes the initial step in the conjugation cascade by forming a thioester bond between the terminal carboxylate of UFM1 (Ubiquitin-fold modifier 1) and a conserved cysteine residue. This ATP-dependent reaction follows a two-step mechanism:

• Adenylation: UBA5 uses ATP to activate UFM1, forming a UFM1-adenylate intermediate

• Thioester formation: The activated UFM1 is transferred to the catalytic cysteine of UBA5

Unlike larger canonical E1 enzymes, UBA5 represents a structurally minimalistic E1 that lacks traditional FCCH (First Catalytic Cysteine Half-domain) or SCCH (Second Catalytic Cysteine Half-domain) domains while maintaining essential catalytic functionality .

What is the genomic location of GK10218 in the D. willistoni genome and how has this been mapped?

The precise genomic location of GK10218 must be understood in the context of recent reassignments of D. willistoni genome scaffolds. A cytological mapping study using in situ hybridization with 22 gene markers revealed that chromosome arms IIL and IIR in D. willistoni correspond to Muller elements B and C, respectively, directly contrasting previous homology assignments .

This finding constitutes a major reassignment of scaffolds to chromosome II arms in D. willistoni. Researchers investigating GK10218 should reference these updated genomic coordinates, as the gene would be located on one of these reassigned scaffolds within the polytene chromosome structure of D. willistoni .

How does the catalytic mechanism of D. willistoni UBA5 differ from other E1 enzymes?

The D. willistoni UBA5 enzyme exhibits several distinctive catalytic features compared to canonical E1 enzymes:

The unique position of the catalytic cysteine near the N-terminus of the α6-helix likely contributes to its nucleophilic character through helix dipole effects. Studies on similar protein structures have shown that cysteines at or near N-terminal positions of α-helices can experience pKa reductions of 0.5-2.1 pH units, enhancing their reactivity .

What structural features are essential for UBA5 function and how can mutations impact its activity?

Several critical structural features determine UBA5 functionality:

• ATP-binding domain: An eight-stranded β-sheet surrounded by seven α-helices that forms the core adenylation domain

• Zinc-binding site: Coordinated by four cysteines with tetrahedral geometry, providing structural stability

• Catalytic cysteine region: Located at the N-terminus of the α6-helix (Cys250 in human UBA5)

• C-terminal domain (CTD): Required for interactions with the E2 enzyme UFC1 and thioester transfer

• Crossover loop regions: The β6–α6 loop is structurally analogous to crossover loops in other E1-like structures

Mutations affecting these regions can significantly impact enzyme function. Recent research on human UBA5 variants associated with developmental and epileptic encephalopathy (DEE44) demonstrated a spectrum of functional impairments across assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation . These variants were classified into mild, intermediate, and severe categories based on their functional effects, providing a framework for understanding structure-function relationships in UBA5 .

What expression systems and purification strategies are most effective for obtaining active recombinant D. willistoni UBA5?

Based on experimental approaches with homologous enzymes, the following protocol is recommended:

Expression Systems:

• Yeast expression systems appear suitable for D. willistoni UBA5 production

• E. coli systems with solubility-enhancing tags may provide alternatives

Construct Optimization:

• Identify and remove disordered regions (N-terminal and C-terminal)

• Preserve the core adenylation domain containing the catalytic cysteine

• Consider that the N-terminal 56 residues may be dispensable (based on human UBA5 studies)

• The CTD can be removed for basic thioester formation assays but is required for E2 transfer studies

Purification Strategy:

• Affinity chromatography (His-tag or GST-tag)

• Ion exchange chromatography

• Size exclusion chromatography

• Maintain reducing conditions throughout purification to protect the catalytic cysteine

Activity Verification:

• Confirm enzymatic activity through ATP-dependent formation of UBA5-UFM1 thioester intermediates

• Use non-reducing SDS-PAGE to visualize the thioester bond formation

How can researchers comprehensively evaluate UBA5 function using in vitro and in vivo approaches?

A multi-faceted approach to UBA5 functional assessment includes:

In Vitro Biochemical Assays:

In Vivo Drosophila-based Assays:

• Viability assessment: Quantification of eclosion rates under UBA5 manipulation

• Developmental timing: Measurement of larval and pupal development periods

• Lifespan analysis: Survival curves under normal and stress conditions

• Locomotor activity: Climbing assays, activity monitoring

• Neurological function: Bang sensitivity tests, seizure susceptibility

A combined approach using both in vitro and in vivo methods provides the most comprehensive evaluation of UBA5 function, as demonstrated in studies of human UBA5 variants where strong correlation was observed between biochemical defects and physiological phenotypes .

How have genomic rearrangements affected the evolutionary context of UBA5 in Drosophila species?

The genomic context of UBA5 in Drosophila must be understood through the lens of significant chromosomal rearrangements during evolution. Recent cytological mapping has revealed that chromosome arms IIL and IIR in D. willistoni correspond to Muller elements B and C, respectively, directly contradicting previous assignments . This finding has important implications:

• While protein function may be conserved, the genomic neighborhood has undergone substantial reorganization

• Such rearrangements potentially impact gene regulation through altered enhancer-promoter interactions

• The reassignment necessitates reevaluation of syntenic relationships between D. willistoni and other Drosophila species

These genomic rearrangements provide a unique opportunity to study how essential enzymatic functions like those of UBA5 are maintained despite significant changes in chromosomal context. The conservation of function despite genomic reorganization suggests robust evolutionary mechanisms for preserving critical cellular pathways .

What insights can comparison between human and D. willistoni UBA5 provide for understanding enzyme evolution?

Comparative analysis of human and D. willistoni UBA5 reveals several evolutionary insights:

• Catalytic mechanism conservation: The unique arrangement of the catalytic cysteine within the adenylation domain (rather than in a separate SCCH domain) appears to be conserved between human and likely Drosophila UBA5, suggesting early evolutionary origin of this mechanism .

• Domain architecture simplification: UBA5 represents a minimalistic E1 enzyme compared to larger canonical E1s, potentially reflecting an ancestral state or specialized adaptation.

• Functional constraints: The position of the catalytic cysteine near the N-terminus of an α-helix is likely constrained by requirements for cysteine activation through helix dipole effects .

• Structural adaptations: The compact structure of UBA5 with its catalytic cysteine in the adenylation domain may represent an evolutionary adaptation to the specific requirements of the UFM1 pathway.

This cross-species comparison provides valuable insights into the evolutionary trajectory of E1 enzymes and how structural variations can accomplish similar catalytic functions through different mechanisms.

How can D. willistoni UBA5 models be used to study human UBA5-associated encephalopathies?

D. willistoni UBA5 provides an excellent model system for investigating human UBA5-associated developmental and epileptic encephalopathy 44 (DEE44) through several approaches:

• Humanized fly models: Following the methodology described for D. melanogaster, humanized D. willistoni flies expressing wild-type or variant human UBA5 can be generated to study phenotypic effects of disease mutations .

• Allelic strength characterization: In vivo Drosophila assays combined with biochemical studies can classify UBA5 variants based on their functional impact, similar to the mild/intermediate/severe classification established for known DEE44 variants .

• Genotype-phenotype correlations: Drosophila models allow researchers to correlate the severity of biochemical defects with physiological outcomes across multiple parameters:

• Compound screening: Humanized fly models can serve as platforms for screening potential therapeutic compounds that might restore UBA5 function or compensate for its deficiency .

What mechanistic insights from UBA5 studies could inform therapeutic approaches for UBA5-related disorders?

Research on UBA5 structure and function provides several potential avenues for therapeutic development:

• Allelic sensitivity targeting: Studies have shown that DEE44 patients typically carry combinations of mild/strong or intermediate/intermediate UBA5 variants, suggesting that complete loss of UBA5 function is likely lethal . Therapies could be tailored to the specific functional deficits of different variant combinations.

• Structure-based drug design: The crystal structure of human UBA5 bound to ATP reveals specific binding pockets that could be targeted by small molecules to enhance residual activity of compromised variants .

• UFMylation pathway modulation: Understanding the entire UFMylation cascade could identify additional targets for intervention beyond UBA5 itself.

• Protein stabilization approaches: For variants that primarily affect protein stability rather than catalytic function, chemical chaperones or proteostasis regulators might be beneficial.

• Compensatory pathway activation: Identification of parallel pathways that could compensate for defects in the UFMylation system might provide alternative therapeutic targets.

The correlation between in vitro biochemical defects and in vivo phenotypes established in Drosophila models provides a robust framework for evaluating the therapeutic potential of interventions targeting specific aspects of UBA5 function .

What are the most effective approaches for studying UBA5-dependent protein UFMylation in cellular contexts?

To investigate UBA5-dependent protein UFMylation, researchers should consider these methodological approaches:

• Proteomic identification of UFMylated substrates:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with immunoprecipitation of UFM1-conjugated proteins

  • Proximity-based labeling methods (BioID, TurboID) with UBA5 as the bait

  • Comparison of UFMylomes between wild-type and UBA5-variant expressing cells

• Live-cell imaging of UFMylation dynamics:

  • Fluorescently tagged UFM1 combined with super-resolution microscopy

  • FRET-based reporters of UBA5-UFM1 interactions

  • Optogenetic control of UBA5 activity to study temporal aspects of UFMylation

• Structural studies of UBA5-substrate interactions:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cryo-EM analysis of UBA5 in complex with UFM1 and target proteins

  • Crosslinking mass spectrometry to identify transient interaction partners

• Genetic manipulation strategies:

  • CRISPR-Cas9 engineered cellular models with UBA5 variants

  • Conditional knockout systems to study acute loss of UBA5 function

  • Tissue-specific manipulation in Drosophila using the GAL4-UAS system

These approaches, when combined, provide comprehensive insights into the cellular roles and dynamics of UBA5-mediated UFMylation under normal and pathological conditions.

How can researchers integrate structural information with functional data to understand UBA5 mechanism?

Integration of structural and functional data requires a multi-dimensional approach:

Data Integration Framework:

Practical Implementation Steps:

• Computational modeling: Use the UBA5 crystal structure as a template to model D. willistoni UBA5, mapping conserved and divergent regions.

• Structure-guided mutagenesis: Design mutations that target specific structural features (e.g., the ATP-binding pocket, catalytic cysteine environment, zinc-binding site) and assess their impact on enzymatic function.

• Molecular dynamics simulations: Analyze the dynamic behavior of wild-type and variant UBA5 to understand how structural perturbations affect conformational flexibility and catalytic activity.

• Transition state modeling: Combine quantum mechanical calculations with structural data to characterize the energy profile of the UBA5 catalytic reaction.

• Network analysis: Map the allosteric networks within UBA5 structure to understand how distal mutations can affect catalytic function through long-range conformational effects.

The human UBA5 crystal structure bound to ATP provides an excellent starting point for such integrative approaches, offering insights into ATP binding, catalytic site geometry, and potential conformational changes during the reaction cycle.

What emerging technologies could advance our understanding of UBA5 function in development and disease?

Several cutting-edge technologies are poised to transform UBA5 research:

• Single-cell multi-omics: Integration of transcriptomics, proteomics, and metabolomics at single-cell resolution to understand cell-type-specific roles of UBA5 during development.

• Spatial transcriptomics/proteomics: Mapping UBA5 expression and activity patterns in developing tissues to identify spatiotemporal regulation of the UFMylation pathway.

• Organoid models: Development of brain organoids from patient-derived iPSCs carrying UBA5 variants to model developmental aspects of UBA5-associated encephalopathies.

• Cryo-electron tomography: Visualization of UBA5 and the UFMylation machinery in their native cellular context.

• AlphaFold-based structural prediction: Leveraging AI-powered structure prediction to model UBA5 complexes with interacting partners and substrates.

• Base editing and prime editing: Precise correction of UBA5 variants in cellular and animal models to establish causality and test therapeutic approaches.

• Tissue-specific CRISPR screens: Identification of genetic modifiers that enhance or suppress UBA5-associated phenotypes in specific tissues.

These technologies, particularly when combined with existing Drosophila models , promise to provide unprecedented insights into UBA5 biology and potential therapeutic targets.

How might cross-species comparative studies of UBA5 enhance our understanding of fundamental UFMylation mechanisms?

Cross-species comparative studies offer unique opportunities to decode the core principles of UFMylation:

• Evolutionary conservation mapping: Systematic comparison of UBA5 sequences and structures across diverse species (from yeast to mammals) can identify absolutely conserved residues that likely play critical roles in fundamental catalytic mechanisms.

• Natural variant analysis: Identification of naturally occurring UBA5 variants across Drosophila species could reveal functionally tolerant regions versus highly constrained domains.

• Substrate conservation: Comparative proteomics to identify conserved UFMylation targets across species would highlight the most ancient and essential functions of this pathway.

• Regulatory network evolution: Analysis of UBA5 regulation across species could reveal how this fundamental pathway has been integrated into different developmental and stress-response programs.

• Domain shuffling and acquisition: Studying the modular nature of UBA5 across species might reveal how this E1 enzyme acquired or lost domains during evolution, potentially informing protein engineering approaches.

The reassignment of D. willistoni genome scaffolds provides a unique opportunity to study how conserved pathways like UFMylation maintain their function despite significant genomic reorganization, offering insights into the evolutionary robustness of essential cellular mechanisms.