Recombinant Neosartorya fumigata Cytoplasmic tRNA 2-thiolation protein 2 (ncs2)

What is the primary function of Neosartorya fumigata Cytoplasmic tRNA 2-thiolation protein 2 (ncs2)?

Neosartorya fumigata Cytoplasmic tRNA 2-thiolation protein 2 (ncs2) is involved in the post-transcriptional modification of transfer RNAs (tRNAs), specifically in the thiolation of uridine residues present at the wobble position in a subset of tRNAs. This protein plays a crucial role in the 2-thiolation of mcm(5)S(2)U at tRNA wobble positions, particularly in tRNA(Lys), tRNA(Glu), and tRNA(Gln). The thiolation process results in enhanced codon reading accuracy during translation . Based on homology to the human CTU2 protein, ncs2 likely forms a heterodimer with CTU1/ATPBD3 that ligates sulfur from thiocarboxylated URM1 onto the uridine of tRNAs at the wobble position .

How does ncs2 contribute to fungal physiology?

The ncs2 protein contributes to fungal physiology through its role in tRNA modification, which directly impacts translational fidelity and efficiency. In Aspergillus fumigatus (closely related to Neosartorya fumigata), disruption of key metabolic pathways can result in significant remodeling of the secondary metabolome, affecting the production of various bioactive compounds . By ensuring accurate tRNA function through proper thiolation, ncs2 likely supports optimal protein synthesis, particularly under stress conditions where translational accuracy becomes critical. This protein may also indirectly influence the production of various secondary metabolites by ensuring proper translation of enzymes involved in their biosynthesis, similar to how disruption of other metabolic genes can lead to widespread metabolomic alterations in A. fumigatus .

What is the relationship between Neosartorya fumigata and Aspergillus fumigatus?

Neosartorya fumigata represents the teleomorphic (sexual) state of Aspergillus fumigatus, which is the anamorphic (asexual) state of the same organism . Taxonomic characterization studies have employed various biochemical and molecular approaches to establish this relationship, including examination of secondary metabolites, ubiquinone systems, isoenzyme patterns, and DNA analysis . Molecular techniques such as restriction fragment length polymorphisms (RFLP) of total DNA, mitochondrial DNA, and ribosomal DNA have been particularly useful in confirming this relationship . When working with ncs2, it's important to recognize that research on A. fumigatus proteins often applies to their N. fumigata counterparts due to this taxonomic relationship.

What are the optimal conditions for recombinant expression of Neosartorya fumigata ncs2?

For recombinant expression of N. fumigata ncs2, E. coli-based expression systems using pET vectors with T7 promoters typically yield good results. Based on protocols for similar proteins, the following conditions are recommended:

• Expression system: E. coli BL21(DE3) or Rosetta(DE3) strains

• Temperature: Induction at lower temperatures (16-18°C) often improves solubility

• Induction: 0.1-0.5 mM IPTG for 16-20 hours

• Media supplements: Addition of rare tRNAs and chaperone co-expression may improve yield

• Buffer conditions: Tris-HCl (pH 7.5-8.0) with 300-500 mM NaCl and 5-10% glycerol

What purification strategies are most effective for isolating recombinant ncs2?

A multi-step purification approach is recommended for isolating high-purity recombinant ncs2:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged ncs2

• Intermediate purification: Ion exchange chromatography (typically anion exchange using Q-Sepharose)

• Polishing step: Size exclusion chromatography for final purification and buffer exchange

The following buffer conditions have been found effective for maintaining ncs2 stability during purification:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 5 mM β-mercaptoethanol

• Elution buffer: Same as lysis buffer but with 250-300 mM imidazole

• Storage buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

Protein should be stored at -80°C, preferably in small aliquots to avoid freeze-thaw cycles that may reduce activity.

How can I assess the functional activity of purified ncs2?

To assess the functional activity of purified ncs2, the following assays can be employed:

• tRNA binding assay: Using electrophoretic mobility shift assay (EMSA) with radiolabeled or fluorescently labeled tRNA substrates

• Thiolation activity assay: In vitro reconstitution of the thiolation pathway with purified CTU1 (ATPBD3), URM1, tRNA substrates, and detection of thiolated uridine using:

  • Mass spectrometry

  • HPLC analysis with thiouridine-specific detection

  • Radiolabeled sulfur incorporation assays

• Heterodimer formation assay: Co-immunoprecipitation or size exclusion chromatography to detect complex formation with CTU1

For all functional assays, controls should include heat-inactivated ncs2 and catalytically inactive mutants (typically mutations in conserved active site residues).

What structural features are essential for ncs2 function?

While the specific structure of N. fumigata ncs2 has not been fully characterized, based on homology to the human CTU2 protein, several domains and motifs are likely critical for its function:

• RNA binding domain: Responsible for recognition and binding of specific tRNA substrates

• CTU1 interaction interface: Mediates heterodimer formation with CTU1/ATPBD3

• Catalytic domain: Contains conserved residues involved in the thiolation reaction

• Zinc-binding motifs: May be present for structural stability

Site-directed mutagenesis of conserved residues combined with functional assays can help identify essential structural features. Typical approaches include alanine scanning of conserved residues followed by binding and activity assays to determine their importance for function.

How does ncs2 interact with its partner proteins in the thiolation pathway?

Based on studies of homologous proteins, ncs2 likely forms a functional complex with other proteins in the thiolation pathway, including:

• CTU1/ATPBD3: Forms a heterodimer with ncs2, essential for tRNA thiolation activity

• URM1: Provides activated sulfur for the thiolation reaction

• Specific tRNAs: Primarily tRNA(Lys), tRNA(Glu), and tRNA(Gln) containing a uridine at the wobble position

The interaction between ncs2 and CTU1 can be studied using:

• Yeast two-hybrid assays

• Co-immunoprecipitation

• Surface plasmon resonance

• Isothermal titration calorimetry

• Hydrogen-deuterium exchange mass spectrometry

These approaches can determine binding affinities, interaction domains, and the dynamics of complex formation in the thiolation pathway.

How conserved is ncs2 across fungal species?

The ncs2 protein is highly conserved across fungal species, reflecting its essential role in tRNA modification. Comparative genomic analysis reveals conservation patterns that can provide insights into functional domains:

The high conservation of the core catalytic domain across distant fungal species underscores its essential function. Phylogenetic analysis can reveal evolutionary relationships and potential adaptive modifications in different fungal lineages.

How does fungal ncs2 differ from its human homolog CTU2?

While both fungal ncs2 and human CTU2 perform similar functions in tRNA thiolation, several key differences exist:

• Sequence divergence: Typically 30-40% sequence identity between fungal ncs2 and human CTU2

• Domain organization: Differences in N- and C-terminal extensions

• Substrate specificity: Potential differences in tRNA recognition patterns

• Regulation: Different regulatory mechanisms controlling expression and activity

• Protein-protein interactions: Variations in interaction partners beyond the core thiolation machinery

These differences can potentially be exploited for the development of selective inhibitors that target fungal ncs2 without affecting human CTU2, which could have implications for antifungal research. Structural biology approaches, including X-ray crystallography or cryo-EM of both proteins, would provide valuable insights into these differences.

What is the significance of ncs2 in Aspergillus/Neosartorya pathogenicity?

The significance of ncs2 in Aspergillus/Neosartorya pathogenicity is linked to its role in translational fidelity, which may impact various virulence factors:

• Stress adaptation: Proper tRNA modification is crucial for accurate translation under stress conditions encountered during host invasion

• Metabolite production: Disruption of tRNA modification pathways can affect secondary metabolite production, similar to how disruption of gliotoxin biosynthesis affects the production of multiple metabolites in A. fumigatus

• Growth rate: Impaired translation may affect fungal growth rate and ability to establish infection

• Protein synthesis fidelity: Essential for producing functional virulence factors

A. fumigatus is responsible for approximately 90% of human Aspergillus infections, making it a significant pathogen, particularly in immunocompromised individuals . Understanding the role of ncs2 in pathogenicity could provide insights into fungal virulence mechanisms and potential intervention strategies.

How might inhibition of ncs2 affect fungal growth and metabolism?

Inhibition of ncs2 would likely disrupt tRNA thiolation, leading to several potential effects on fungal growth and metabolism:

• Translational errors: Increased mistranslation due to impaired codon recognition at the wobble position

• Stress sensitivity: Reduced ability to adapt to environmental stresses due to compromised protein synthesis

• Metabolic disruption: Altered production of secondary metabolites and enzymes

• Growth inhibition: Potential reduction in growth rate, especially under stress conditions

Based on studies of metabolic pathway disruption in A. fumigatus, inhibition of ncs2 could lead to widespread proteomic alterations. For example, disruption of gliotoxin biosynthesis in A. fumigatus resulted in significant changes to 260 proteins, including those involved in secondary metabolism, amino acid biosynthesis, and stress response . Similar widespread effects might be expected from ncs2 inhibition, potentially affecting multiple biosynthetic gene clusters and their associated metabolites.

How can CRISPR-Cas9 technology be optimized for studying ncs2 function in Neosartorya fumigata?

Optimizing CRISPR-Cas9 for studying ncs2 function in N. fumigata requires addressing several challenges specific to filamentous fungi:

• Delivery method:

  • Protoplast transformation with ribonucleoprotein (RNP) complexes

  • Agrobacterium-mediated transformation for difficult-to-transform strains

• Guide RNA design:

  • Target unique sequences with minimal off-target potential

  • Verify specificity using fungal-specific CRISPR design tools

  • Consider GC content and secondary structure

• Homology-directed repair (HDR) template design:

  • 1-2 kb homology arms for efficient integration

  • Include selectable markers (hygromycin or pyrithiamine resistance)

  • Consider silent mutations in PAM sites to prevent re-cutting

• Verification strategies:

  • PCR screening with primers spanning integration junctions

  • Whole-genome sequencing to detect off-target effects

  • RT-qPCR and Western blotting to confirm knockout/knockdown

  • Functional assays to assess tRNA thiolation levels

For more sophisticated applications, consider inducible or conditional systems that allow temporal control of ncs2 expression, which can be particularly useful for studying essential genes where complete knockout may be lethal.

What are the challenges in crystallizing ncs2 for structural studies, and how can they be overcome?

Crystallizing fungal ncs2 presents several challenges typical of eukaryotic proteins involved in complex formation:

• Protein stability issues:

  • Solution: Screen multiple buffer conditions (pH 6.5-8.5, various salt concentrations)

  • Add stabilizing agents (glycerol, trehalose, specific metal ions)

  • Consider truncated constructs that remove flexible regions

• Heterogeneity:

  • Solution: Employ size-exclusion chromatography immediately before crystallization

  • Use dynamic light scattering to confirm monodispersity

  • Consider limited proteolysis to identify stable domains

• Low solubility:

  • Solution: Test fusion partners (MBP, SUMO, Trx) to enhance solubility

  • Screen solubility-enhancing additives (arginine, glutamate, non-detergent sulfobetaines)

• Complex formation:

  • Solution: Co-express with binding partners (CTU1/ATPBD3)

  • Co-crystallize with substrate tRNAs or substrate analogs

• Crystallization screening:

  • Initial approach: Commercial sparse matrix screens at multiple temperatures (4°C, 16°C, 20°C)

  • Optimization: Fine grid screens around initial hits

  • Advanced techniques: Seeding, counter-diffusion, lipidic cubic phase for membrane-associated regions

For difficult targets, consider alternative structural biology approaches such as cryo-electron microscopy, which has become increasingly viable for proteins >100 kDa, especially when in complex with partners like tRNAs.

Why might recombinant ncs2 show low enzymatic activity despite high purity?

Several factors can contribute to low enzymatic activity of recombinant ncs2 despite high purity:

• Misfolding during expression:

  • Solution: Try different expression hosts (yeast systems instead of E. coli)

  • Lower induction temperature (16°C overnight instead of 37°C)

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Missing cofactors or post-translational modifications:

  • Solution: Add potential cofactors to reaction buffer (zinc, iron, ATP)

  • Consider expression in eukaryotic systems that provide appropriate modifications

• Absence of essential binding partners:

  • Solution: Co-express or add purified CTU1/ATPBD3 protein

  • Reconstitute complete thiolation pathway with URM1 and activation enzymes

• Buffer incompatibility:

  • Solution: Screen various buffer conditions (pH, salt concentrations, reducing agents)

  • Test different metal ions that might be required for catalysis

• Protein oxidation:

  • Solution: Increase concentration of reducing agents (DTT, TCEP)

  • Perform purification and assays under anaerobic conditions

If issues persist, limited proteolysis followed by mass spectrometry can help identify stable domains that might be more amenable to functional studies, potentially revealing if specific regions are improperly folded.

How can I differentiate between direct and indirect effects when studying ncs2 knockout phenotypes?

Differentiating between direct and indirect effects in ncs2 knockout studies requires a systematic approach:

• Complementation studies:

  • Reintroduce wild-type ncs2 to verify phenotype rescue

  • Use catalytically inactive mutants to determine if phenotypes are activity-dependent

• Temporal analysis:

  • Use time-course experiments to identify primary (early) versus secondary (late) effects

  • Employ inducible systems to observe immediate consequences of ncs2 depletion

• Specific activity measurements:

  • Directly measure tRNA thiolation levels using mass spectrometry

  • Quantify thiolated versus non-thiolated tRNA ratios

• Targeted versus global analysis:

  • Compare transcriptome, proteome, and metabolome data to identify patterns

  • Look for enrichment of specific pathways that might explain indirect effects

• Epistasis analysis:

  • Combine ncs2 knockout with knockouts of downstream factors

  • If phenotypes are suppressed, this suggests indirect effects through those pathways

Similar approaches have been used in A. fumigatus studies, where disruption of gliotoxin biosynthesis led to widespread metabolomic and proteomic changes . By employing complementary strains and detailed time-course analyses, researchers were able to distinguish primary effects from secondary adaptations.

What are promising approaches for developing selective inhibitors of fungal ncs2?

Developing selective inhibitors of fungal ncs2 presents several promising research avenues:

• Structure-based drug design:

  • Comparative modeling of fungal ncs2 and human CTU2

  • Virtual screening of compound libraries against fungal-specific binding pockets

  • Fragment-based screening to identify starting scaffolds

• High-throughput screening approaches:

  • Develop fluorescence-based assays for tRNA thiolation

  • Screen natural product libraries, particularly fungal-derived compounds

  • Repurpose existing compound libraries with known antifungal activity

• Peptide-based inhibitors:

  • Design peptides that mimic CTU1-binding interface

  • Develop stapled peptides for improved stability and cell penetration

• RNA-based strategies:

  • Design RNA aptamers that specifically bind fungal ncs2

  • Develop antisense oligonucleotides targeting ncs2 mRNA

• Allosteric inhibitors:

  • Target fungal-specific regulatory sites rather than conserved active sites

  • Exploit differences in protein dynamics between fungal and human proteins

Computational approaches including molecular dynamics simulations can help identify transient pockets or conformational states unique to fungal ncs2 that might not be apparent in static structures, providing additional targeting opportunities.

How might systems biology approaches enhance our understanding of ncs2 function within the broader context of fungal metabolism?

Systems biology approaches offer powerful frameworks for understanding ncs2 within fungal metabolism:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from ncs2 mutants

  • Use network analysis to identify affected pathways and regulatory hubs

  • Similar approaches revealed that disruption of gliotoxin biosynthesis in A. fumigatus affected 260 proteins across multiple pathways

• Flux analysis:

  • Apply metabolic flux analysis using stable isotope labeling

  • Track changes in metabolic pathways resulting from altered translation fidelity

• Protein-protein interaction networks:

  • Use proximity labeling (BioID, APEX) to identify the ncs2 interactome

  • Construct interaction networks to place ncs2 in cellular context

• Mathematical modeling:

  • Develop predictive models of how tRNA modification affects translation rate and accuracy

  • Simulate effects of ncs2 inhibition on protein synthesis and metabolic pathways

• Comparative systems analysis:

  • Compare system-wide effects of ncs2 disruption across multiple fungal species

  • Identify conserved versus species-specific responses

These approaches can reveal how tRNA thiolation connects to broader aspects of fungal physiology, potentially uncovering unexpected connections to virulence, stress response, and secondary metabolism regulation.