Recombinant Neosartorya fumigata tRNA (adenine (58)-N (1))-methyltransferase catalytic subunit trm61 (trm61), partial

What is the basic structure of the TRM61 protein in Neosartorya fumigata?

The TRM61 protein in N. fumigata, similar to its homolog in Saccharomyces cerevisiae, consists of an N-terminal β-barrel domain connected to a C-terminal Rossmann-fold domain. The Rossmann-fold domain is characteristic of methyltransferases and contains the S-adenosyl-L-methionine (SAM) binding pocket essential for catalytic activity. Crystal structure analysis of homologous TRM61 proteins reveals that TRM61 functions as part of a heterotetramer, forming a complex with its non-catalytic partner TRM6, with two TRM6-TRM61 heterodimers assembling together . The catalytic subunit TRM61 contains conserved motifs necessary for binding the methyl donor SAM, which the TRM6 subunit lacks.

How does the TRM61-TRM6 complex catalyze tRNA m1A58 methylation?

The TRM61-TRM6 complex catalyzes the N1-methylation of adenine at position 58 (m1A58) in tRNA molecules. TRM61 functions as the catalytic subunit containing the SAM binding pocket, while TRM6 lacks the conserved motifs for SAM binding but cooperates with TRM61 to form an L-shaped tRNA binding region . During the methylation process, SAM donates a methyl group to the N1 position of adenine at position 58 of the tRNA substrate. This methylation is particularly important for maintaining the stability of initiator methionine tRNA (tRNAi Met). The reaction involves positioning of the target adenine in the active site of TRM61, followed by nucleophilic attack on the methyl group of SAM.

Why is the m1A58 modification important in fungal biology?

The m1A58 modification plays a critical role in fungal biology by ensuring tRNA structural stability and proper function during protein translation. This modification is particularly vital for the stability of initiator methionine tRNA (tRNAi Met) . In eukaryotes, disruption of this modification can lead to degradation of hypomodified tRNAi Met, resulting in translation initiation defects. In fungal pathogens like N. fumigata, proper tRNA modification is essential for protein synthesis required during infection processes, stress response, and cellular adaptation. Studies on homologous systems indicate that loss of this modification can significantly impact cell growth and viability, suggesting it may be a potential target for antifungal development.

What expression systems are optimal for producing recombinant N. fumigata TRM61?

For recombinant expression of N. fumigata TRM61, several expression systems can be employed based on research needs:

For most structural and biochemical studies, E. coli expression with an N-terminal His-tag followed by TEV protease cleavage site has been successful for homologous proteins. When expressing in E. coli, co-expression with the TRM6 subunit is often necessary to obtain soluble and active TRM61, as the heterodimer formation appears critical for proper folding and stability .

What are the most effective purification strategies for recombinant TRM61-TRM6 complex?

Purification of the TRM61-TRM6 complex typically involves a multi-step approach:

• Affinity Chromatography: Using a His-tagged construct (typically on either TRM61 or TRM6) followed by immobilized metal affinity chromatography (IMAC) using Ni-NTA resin.

• Ion Exchange Chromatography: Following IMAC, ion exchange (typically Q-Sepharose for anion exchange) helps remove contaminants and nucleic acids that may co-purify.

• Size Exclusion Chromatography: A final gel filtration step (often using Superdex 200) isolates the intact heterotetramer complex and removes aggregates.

For optimal results, purification buffers typically contain:

• 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-300 mM NaCl (higher during initial steps, lower during ion exchange)

• 5-10% glycerol for stability

• 1-5 mM DTT or 0.5-2 mM TCEP to maintain reduced cysteines

• Protease inhibitors during initial extraction

Protein purity should be assessed by SDS-PAGE, and activity can be confirmed using methyltransferase assays with appropriate tRNA substrates. Western blotting with antibodies against both subunits can verify the presence of both proteins in the purified complex.

How can you verify the proper folding and assembly of recombinant TRM61-TRM6 complex?

Verification of proper folding and assembly of the recombinant TRM61-TRM6 complex involves several analytical techniques:

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This technique provides accurate molecular weight determination of the complex in solution, confirming the expected heterotetramer assembly (two TRM61-TRM6 heterodimers) .

• Circular Dichroism (CD) Spectroscopy: CD analysis in the far-UV range (190-260 nm) provides information about secondary structure content, which can be compared to expected values based on homologous structures.

• Thermal Shift Assay (Differential Scanning Fluorimetry): This technique measures protein thermal stability, which can indicate proper folding. Well-folded complexes typically exhibit cooperative unfolding and higher melting temperatures.

• Limited Proteolysis: Controlled digestion with proteases can reveal structural domains and indicate proper folding as correctly folded proteins show resistance to digestion at domain boundaries.

• SAM Binding Assay: Monitoring the binding of the cofactor S-adenosyl-L-methionine using techniques like isothermal titration calorimetry (ITC) or differential scanning fluorimetry with SAM can confirm proper folding of the catalytic pocket in TRM61.

• Activity Assays: Ultimately, the most definitive verification comes from enzymatic activity assays measuring the transfer of methyl groups to appropriate tRNA substrates.

What crystallization conditions have been successful for TRM61-TRM6 complexes?

Based on successful crystallization of homologous TRM61-TRM6 complexes, the following conditions have proven effective:

For co-crystallization with SAM, pre-incubation with 2-5 mM SAM before setting up crystallization trials has been successful . Microseeding from initial crystal hits can significantly improve crystal quality. Based on structures of homologous complexes, crystals have been obtained in various space groups, with P212121 being common for the heterotetramer.

The addition of tRNA substrate has been challenging for crystallization, potentially due to conformational flexibility. In these cases, limited proteolysis to remove flexible regions or the use of tRNA fragments may facilitate crystallization of substrate-bound complexes.

What structural techniques beyond X-ray crystallography are useful for studying TRM61-TRM6 interactions?

Several complementary structural techniques can provide valuable insights into TRM61-TRM6 interactions:

These techniques can be particularly valuable for understanding the dynamic aspects of TRM61-TRM6 function that may not be evident from static crystal structures.

What are the most reliable methods for measuring TRM61 methyltransferase activity?

Several reliable methods can be employed to measure TRM61 methyltransferase activity:

• Radiometric Assay: This classic approach uses S-adenosyl-L-[methyl-³H]methionine as the methyl donor. The reaction mixture contains:

  • Purified TRM61-TRM6 complex (50-200 nM)

  • In vitro transcribed or purified tRNA substrate (0.5-5 μM)

  • Radiolabeled SAM (typically 1-5 μM, 10 Ci/mmol)

  • Buffer: 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT

  After incubation (30-60 minutes at 30-37°C), tRNA is precipitated with trichloroacetic acid (TCA) on filter papers, washed, and counted using scintillation counting. This method provides high sensitivity but requires radioactive material handling.

• HPLC-Based Detection: After the methylation reaction with non-radioactive SAM, tRNA is digested to nucleosides using nuclease P1 and alkaline phosphatase. The resulting nucleosides are separated by HPLC, and m¹A can be detected and quantified by UV absorbance or mass spectrometry.

• Antibody-Based Detection: Using antibodies specific for m¹A, modified tRNAs can be detected via immunoblotting or ELISA-based approaches.

• Mass Spectrometry: LC-MS/MS analysis of digested tRNA provides precise identification and quantification of modified nucleosides, allowing for comparison of m¹A58 levels between different samples.

• SAM to SAH Conversion Assay: Commercial kits measuring the conversion of SAM to S-adenosyl-L-homocysteine (SAH) can indirectly measure methyltransferase activity through coupled enzymatic reactions that produce a fluorescent or colorimetric output.

For kinetic analysis, it's recommended to use the radiometric approach or HPLC-MS/MS methods, varying either substrate concentration (0.1-10 μM tRNA) or enzyme concentration to determine Km, Vmax, and kcat values.

How can you prepare suitable tRNA substrates for TRM61 activity assays?

Preparation of suitable tRNA substrates for TRM61 activity assays can be accomplished through several methods:

• In Vitro Transcription:

  • Clone the target tRNA gene (particularly tRNAi Met) into a vector containing a T7 promoter

  • Linearize the plasmid at the 3' end of the tRNA sequence

  • Perform in vitro transcription using T7 RNA polymerase

  • Purify the transcribed tRNA using denaturing PAGE (8-10% polyacrylamide, 8M urea)

  • Elute tRNA from gel slices and refold by heating to 65°C followed by slow cooling in the presence of 1-5 mM MgCl₂

• Isolation from Organisms Lacking m1A58 Modification:

  • Extract total tRNA from trm61 or trm6 deletion mutants (in S. cerevisiae or other model organisms)

  • Enrich for specific tRNAs using oligonucleotide-directed affinity purification or biotinylated complementary oligonucleotides

  • Verify the absence of m1A58 modification by HPLC or mass spectrometry analysis

• Chemical Synthesis:

  • For shorter tRNA fragments containing the TΨC loop (where A58 is located), chemical synthesis can be employed

  • These synthetic fragments (typically 15-30 nucleotides) can sometimes serve as minimal substrates for the enzyme

For optimal enzyme activity, the tRNA substrate should be properly folded. This can be achieved by heating the tRNA to 65-80°C in water, followed by gradual cooling in the presence of buffer containing 5-10 mM MgCl₂. The quality of the tRNA substrate can be assessed by native PAGE and thermal melting profiles.

It's important to note that the TRM61-TRM6 complex typically shows preference for certain tRNA species, with tRNAi Met often being the preferred substrate in eukaryotic systems . Testing multiple tRNA species might be necessary to identify optimal substrates for the N. fumigata enzyme.

How do mutations in conserved motifs affect TRM61 catalytic activity?

Mutations in conserved motifs of TRM61 can significantly impact its catalytic activity. Based on studies of homologous enzymes, the following effects have been observed:

Specific mutations that have been characterized in homologous systems include:

• Mutations in the SAM-binding pocket that disrupt hydrogen bonding with the methionine moiety of SAM

• Alterations to the catalytic aspartate that positions the target adenine

• Disruption of the L-shaped tRNA binding region formed by TRM6-TRM61 cooperation

These mutations typically result in reduced catalytic efficiency (lower kcat/Km values), rather than complete loss of SAM binding. This suggests that proper positioning of both SAM and the target adenine is critical for the methyl transfer reaction. Additionally, mutations at the TRM6-TRM61 interface can destabilize the complex, indirectly reducing catalytic activity by impairing substrate binding.

How does TRM61 function differ between Neosartorya fumigata and related fungal species?

The TRM61 function shows both conservation and species-specific adaptations across fungal species:

In N. fumigata, TRM61 appears to be integrated into stress response pathways that are particularly important during host invasion and adaptation to the mammalian environment. While the core catalytic mechanism is conserved across species, N. fumigata TRM61 shows adaptations potentially linked to its pathogenic lifestyle, including potential connections to secondary metabolism regulation pathways .

What phenotypes are associated with TRM61 deficiency in pathogenic fungi?

TRM61 deficiency in pathogenic fungi leads to several significant phenotypes:

• Growth and Viability Effects:

  • Severe growth defects or lethality in most pathogenic fungi

  • Temperature-sensitive growth, particularly at host body temperature (37°C)

  • Cell wall integrity defects and abnormal morphology

• tRNA Processing and Stability:

  • Reduced levels of mature tRNAi Met due to degradation of hypomodified molecules

  • Global reduction in translation initiation efficiency

  • Activation of cellular stress response pathways related to protein synthesis

• Virulence Impacts:

  • Significantly attenuated virulence in animal infection models

  • Reduced ability to adapt to host environmental conditions

  • Compromised stress response, particularly to oxidative and temperature stress encountered during host infection

• Drug Susceptibility:

  • Increased sensitivity to antifungal agents, particularly those targeting protein synthesis

  • Altered cell wall composition affecting susceptibility to echinocandins

  • Synergistic effects when combined with sub-inhibitory concentrations of translation inhibitors

In Aspergillus species like N. fumigata, TRM61 deficiency also appears to affect secondary metabolism pathways, which may be linked to altered expression of biosynthetic gene clusters involved in the production of mycotoxins and virulence factors . The interconnection between tRNA modification and secondary metabolism regulation represents an emerging area of research in fungal pathogenesis.

How does TRM61 activity correlate with invasive fungal infection outcomes?

TRM61 activity shows important correlations with invasive fungal infection outcomes:

• Infection Establishment and Progression:

  • Optimal TRM61 activity is crucial during the early stages of invasive aspergillosis

  • The enzyme's function becomes particularly important under host stress conditions like elevated temperature and oxidative environments

  • Full TRM61 activity correlates with more rapid progression of invasive disease in immunocompromised models

• Host-Pathogen Interface Dynamics:

  • TRM61-mediated tRNA modifications support rapid protein synthesis required during host adaptation

  • This enables timely expression of immune evasion factors and stress response proteins

  • Cases of chronic aspergillosis, such as those caused by N. udagawae, may show altered patterns of tRNA modification enzyme expression compared to acute A. fumigatus infections

• Clinical Correlations:

  • In clinical isolates from cases of invasive aspergillosis, TRM61 expression levels tend to be elevated compared to environmental isolates

  • Strains with naturally occurring TRM61 variants show differences in virulence potential

  • In chronic granulomatous disease patients with aspergillosis, fungal isolates may exhibit adaptations in tRNA modification patterns for long-term persistence

The duration of N. udagawae infections (median 35 weeks) compared to A. fumigatus sensu stricto infections (median 5.5 weeks) suggests potential differences in translational regulation strategies between these species . The ability of N. udagawae to establish chronic infection across anatomical planes may involve distinctive patterns of tRNA modification enzyme activity, allowing for persistent but slower growth in host tissues.

How does the structure of fungal TRM61 compare with bacterial and archaeal homologs?

The structure of fungal TRM61 shows significant differences compared to bacterial and archaeal homologs:

The most striking difference is the requirement for the non-catalytic TRM6 subunit in eukaryotic systems, which is absent in bacteria and archaea. This additional subunit in fungi provides extended RNA binding surfaces and regulatory potential not present in prokaryotic systems . The eukaryotic heterotetramer assembly allows for allosteric regulation and potentially more sophisticated substrate selection than the simpler homotetrameric bacterial and archaeal enzymes.

Structurally, while all these enzymes contain the core Rossmann fold methyltransferase domain, the fungal enzyme has evolved additional elements that contribute to its specificity and regulatory potential. These structural differences could potentially be exploited for the development of antifungal compounds that selectively target the fungal enzyme without affecting human homologs.

What functional differences exist between TRM61 in fungi and humans?

Several important functional differences exist between fungal and human TRM61:

• Substrate Specificity:

  • Fungal TRM61: Shows strong preference for tRNAi Met with secondary activity on a limited set of other tRNAs

  • Human TRM61: Broader substrate range including both initiator and elongator tRNAs, with evidence for activity on certain mRNAs as well

• Cellular Localization:

  • Fungal TRM61: Primarily nuclear localization with evidence for some cytoplasmic activity

  • Human TRM61: Nuclear localization with specific targeting to nucleoli, suggesting association with rRNA processing machinery

• Regulation and Interaction Networks:

  • Fungal TRM61: In N. fumigata, potential integration with secondary metabolism regulatory networks

  • Human TRM61: Interactions with RNA polymerase III machinery and potential roles in cancer cell metabolism

• Physiological Consequences of Deficiency:

  • Fungal TRM61: Often lethal or severely growth-inhibiting

  • Human TRM61: Linked to developmental defects and certain cancers; siRNA-mediated depletion in mammalian cells causes significant effects on cell growth, cell death, and tRNAi Met levels

• Post-translational Modifications:

  • Fungal TRM61: Limited evidence for regulation by phosphorylation

  • Human TRM61: Multiple phosphorylation and ubiquitination sites with evidence for cell-cycle dependent regulation

These functional differences, particularly in substrate recognition and regulatory networks, provide potential targets for selective inhibition of the fungal enzyme. The broader substrate range of human TRM61 may also explain why it appears to be more tightly regulated than its fungal counterpart, potentially as a means to control its activity on a wider array of cellular RNAs.

How have evolutionary analyses informed our understanding of TRM61 function?

Evolutionary analyses have provided several key insights into TRM61 function:

• Evolutionary Origin and Divergence:

  • TRM61 evolved from a common ancestor with bacterial TrmI

  • The split between TRM61 (catalytic) and TRM6 (non-catalytic) occurred early in eukaryotic evolution

  • TRM6 appears to have originated from a gene duplication of TRM61 followed by loss of catalytic residues while retaining RNA binding capabilities

• Conservation Patterns:

  • The SAM-binding pocket shows the highest sequence conservation across species

  • The TRM6-TRM61 interface is highly conserved in eukaryotes, underscoring its importance

  • Surface loops show the greatest variability, suggesting species-specific adaptations for regulation or substrate recognition

• Coevolution with tRNA Substrates:

  • Correlation between TRM61 sequence features and tRNA gene content in different organisms

  • Evidence for coevolution with specific features of initiator tRNA

  • Adaptations to organism-specific tRNA modification patterns

• Selective Pressure Analysis:

  • Stronger purifying selection on catalytic core compared to peripheral regions

  • Evidence for positive selection at the RNA binding interface in some fungal lineages

  • Different selective pressures between pathogenic and non-pathogenic fungi suggesting adaptation to host environments

• Horizontal Gene Transfer:

  • No significant evidence for horizontal transfer of TRM61 between distant fungal lineages

  • Vertical inheritance with sequence divergence proportional to phylogenetic distance

These evolutionary insights have practical applications for targeting TRM61 in pathogenic fungi. By identifying fungal-specific features that emerged during evolution, researchers can develop inhibitors that exploit these differences. Additionally, understanding the coevolution of TRM61 with tRNA substrates helps explain substrate preference patterns observed across species.

What are common challenges in expressing and purifying active recombinant N. fumigata TRM61?

Researchers commonly encounter several challenges when expressing and purifying active recombinant N. fumigata TRM61:

• Solubility Issues:

  • Challenge: TRM61 often forms inclusion bodies when expressed alone in E. coli

  • Solution: Co-expression with TRM6 significantly improves solubility; use lower induction temperatures (16-18°C); employ solubility-enhancing tags like MBP or SUMO

• Stability Problems:

  • Challenge: Purified protein shows aggregation and activity loss during storage

  • Solution: Include 5-10% glycerol and 1-5 mM DTT or TCEP in storage buffers; store at high concentration (>1 mg/ml); avoid freeze-thaw cycles by flash-freezing aliquots in liquid nitrogen

• Heterogeneity in Preparations:

  • Challenge: Variable stoichiometry of TRM61-TRM6 complex

  • Solution: Use tandem affinity purification with tags on both subunits; verify complex formation by size exclusion chromatography; optimize salt concentration during purification to maintain complex integrity

• Nucleic Acid Contamination:

  • Challenge: Co-purification of E. coli RNA that interferes with activity assays

  • Solution: Include high salt washes (500 mM NaCl) during initial purification steps; treat with RNase A followed by heparin chromatography; use polyethyleneimine precipitation before initial chromatography

• Low Activity of Purified Enzyme:

  • Challenge: Enzyme shows significantly lower activity than expected

  • Solution: Verify proper folding by circular dichroism; ensure SAM integrity in assays (SAM degrades easily); optimize buffer conditions (particularly Mg²⁺ concentration); ensure tRNA substrates are properly folded

• Non-specific Aggregation During Concentration:

  • Challenge: Protein aggregates during concentration steps

  • Solution: Use spin concentrators with regenerated cellulose membranes; concentrate in the presence of higher glycerol (10%); perform concentration at 4°C with gentle intermittent mixing

Implementing these solutions has been shown to significantly improve the quality and yield of active TRM61-TRM6 complex for both structural and functional studies.

How can activity assays for TRM61 be optimized for higher sensitivity and reproducibility?

Optimizing TRM61 activity assays for higher sensitivity and reproducibility involves several key strategies:

• Buffer Optimization:

  • Systematically test buffer components:

    • pH range: 7.0-8.5 (typically optimal around pH 8.0)

    • Salt concentration: 50-200 mM (typically 100 mM NaCl or KCl)

    • Divalent cations: 1-10 mM MgCl₂ or MnCl₂

    • Reducing agents: 1-5 mM DTT or 0.5-2 mM TCEP

  • Include stabilizers like 5% glycerol and 0.01% Triton X-100 to prevent non-specific adsorption

• Substrate Preparation Refinement:

  • Ensure tRNA is properly folded by heating to 65°C followed by slow cooling

  • Verify tRNA integrity by native PAGE before each assay

  • For in vitro transcribed tRNA, remove 5'-triphosphate by phosphatase treatment to eliminate potential inhibitory effects

• Reaction Condition Optimization:

  • Temperature: Test range from 25-37°C (balancing enzyme activity and stability)

  • Time course: Establish linear range of activity (typically 5-30 minutes)

  • Enzyme concentration: Titrate to establish linear response range

  • Use internal controls: Include standardized enzyme preparation in each assay batch

• Detection Method Improvements:

  • For radiometric assays:

    • Increase specific activity of [³H-methyl]-SAM (15-80 Ci/mmol)

    • Implement filter-binding on positively charged membranes rather than precipitation

    • Use scintillation proximity assay (SPA) beads for higher throughput

  • For mass spectrometry detection:

    • Optimize digestion conditions for complete nucleoside release

    • Use isotopically labeled internal standards for quantification

    • Implement multiple reaction monitoring (MRM) for improved sensitivity

• Quality Control Measures:

  • Prepare large batches of substrate and store as single-use aliquots

  • Include positive controls (known active methyltransferase) and negative controls (heat-inactivated enzyme)

  • Implement statistical quality control charts to monitor assay performance over time

By systematically implementing these optimizations, researchers can achieve coefficient of variation (CV) values below 10% and detection limits in the low nanomolar range for TRM61 activity assays.

What strategies can overcome difficulties in crystallizing the TRM61-TRM6 complex with tRNA?

Crystallizing the TRM61-TRM6 complex with tRNA presents significant challenges. The following strategies can help overcome these difficulties:

• Construct Engineering:

  • Create truncated constructs removing flexible regions identified by limited proteolysis

  • Design constructs based on hydrogen-deuterium exchange mass spectrometry (HDX-MS) data to identify stable core regions

  • Create chimeric constructs incorporating stable domains from homologous proteins with known crystal structures

  • Introduce surface entropy reduction mutations (replacing high-entropy residues like Lys/Glu/Gln with Ala) based on computational predictions

• tRNA Substrate Modifications:

  • Use tRNA variants with stabilizing modifications in regions not involved in TRM61-TRM6 binding

  • Generate minimal tRNA constructs focusing on the TΨC loop containing A58

  • Introduce base modifications that lock the tRNA in a single conformation

  • Use 2'-O-methyl modifications at specific positions to reduce conformational flexibility

• Crystallization Methodologies:

  • Implement microseeding from initial hits to improve crystal quality

  • Use streak seeding from related complex crystals (e.g., SAM-bound complex crystals)

  • Explore lipidic cubic phase (LCP) or bicelle crystallization methods

  • Employ counter-diffusion crystallization in capillaries for slower, more ordered crystal growth

• Complex Stabilization:

  • Use chemical crosslinking (e.g., glutaraldehyde, EDC/NHS) with careful optimization to maintain activity

  • Incorporate non-hydrolyzable SAM analogs to stabilize the active site

  • Test RNA methylation inhibitors that can lock the complex in a specific conformation

  • Create disulfide-stabilized complexes by introducing cysteine pairs at the protein-RNA interface

• Alternative Approaches:

  • Consider crystallizing subcomplexes if the full complex proves recalcitrant

  • Use antibody-fragment (Fab) mediated crystallization to provide additional crystal contacts

  • Employ cryo-electron microscopy as an alternative approach for structural determination

  • Implement integrative structural biology combining lower-resolution techniques (SAXS, cryo-EM) with high-resolution structures of components

These strategies have proven effective for similar challenging RNA-protein complexes and can be adapted for the TRM61-TRM6-tRNA system. Successful implementation may require combining multiple approaches simultaneously.

What are the emerging research directions in TRM61 biology and function?

Several exciting research directions are emerging in TRM61 biology and function:

• Regulatory Roles Beyond tRNA Modification:

  • Investigation of TRM61 moonlighting functions in transcriptional regulation

  • Exploration of potential roles in modifying mRNAs or other non-tRNA substrates

  • Examination of connections between TRM61 and fungal secondary metabolism regulation

  • Potential involvement in stress granule formation during cellular stress response

• TRM61 in Fungal Pathogenesis Networks:

  • Integration of TRM61 activity with virulence factor expression networks

  • Role in regulating stress adaptation during host invasion

  • Connection to antifungal resistance mechanisms

  • Potential as a biomarker for invasive fungal infection progression

• Structural and Mechanistic Advances:

  • Application of cryo-EM to visualize the complete catalytic cycle

  • Time-resolved structural studies to capture transition states

  • Computational modeling of the methylation reaction mechanism

  • Investigation of allosteric regulation within the heterotetramer complex

• Therapeutic Target Development:

  • Fragment-based drug discovery targeting TRM61-specific pockets

  • Development of suicide inhibitors exploiting the catalytic mechanism

  • Design of molecules disrupting TRM61-TRM6 interface

  • Creation of fungal-specific inhibitors exploiting structural differences from human homologs

• Systems Biology Approaches:

  • Global analysis of the tRNA modificome in response to TRM61 perturbation

  • Network analysis of TRM61 genetic and physical interactions

  • Integration of transcriptomics, proteomics, and metabolomics data to understand downstream effects of TRM61 dysfunction

  • Evolutionary analysis across fungal pathogens to identify adaptive patterns

These emerging research directions promise to expand our understanding of TRM61 beyond its canonical role in tRNA modification and may reveal novel therapeutic opportunities for treating invasive fungal infections.

What methodological advances would accelerate TRM61 research?

Several methodological advances would significantly accelerate TRM61 research:

• Improved Genetic Manipulation:

  • Development of conditional TRM61 expression systems for essential gene studies

  • CRISPR-Cas9 based methods for rapid generation of point mutations in TRM61

  • Creation of fungal strains with tagged endogenous TRM61 for in vivo studies

  • Implementation of auxin-inducible degron systems for rapid protein depletion

• Advanced Structural Analysis:

  • Time-resolved crystallography to capture catalytic intermediates

  • High-resolution cryo-EM methods for visualizing TRM61-TRM6-tRNA complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflows optimized for RNA-protein complexes

  • Integrative structural biology platforms combining multiple data types

• High-Throughput Activity Assays:

  • Development of fluorescence-based real-time methyltransferase assays

  • Adaptation of methods to microplate format for inhibitor screening

  • Label-free detection methods using surface plasmon resonance (SPR) or bio-layer interferometry

  • Cell-based reporter systems for monitoring TRM61 activity in vivo

• RNA Modification Analysis:

  • Simplified protocols for tRNA isolation with preserved modifications

  • Direct nanopore sequencing of tRNAs to identify modification patterns

  • Antibody-based enrichment methods for m1A-containing tRNAs

  • Improved mass spectrometry methods for quantitative analysis of modified nucleosides

• Computational Tools:

  • Machine learning approaches for predicting TRM61 substrates

  • Molecular dynamics simulations optimized for tRNA-protein complexes

  • Bioinformatic pipelines for analyzing evolutionary patterns in tRNA modification enzymes

  • Systems biology frameworks for modeling the impact of tRNA modifications on translation

• Chemical Biology Approaches:

  • Development of cell-permeable SAM analogs for metabolic labeling

  • Photo-crosslinkable tRNA substrates for capturing transient interactions

  • Activity-based probes for visualizing active TRM61 in cellular contexts

  • Chemically stabilized tRNA mimics for structural studies

Implementation of these methodological advances would address current bottlenecks in TRM61 research, enabling more rapid progress in understanding both fundamental aspects of tRNA biology and potential therapeutic applications.

How might inhibitors of TRM61 be developed as potential antifungal agents?

Development of TRM61 inhibitors as potential antifungal agents could follow several strategic approaches:

• Structure-Based Drug Design:

  • Target the SAM binding pocket with competitive inhibitors

  • Design bisubstrate analogs linking SAM-like and adenine-like moieties

  • Exploit the TRM61-TRM6 interface with protein-protein interaction disruptors

  • Develop allosteric inhibitors targeting fungal-specific regulatory sites

• Phenotypic Screening Approaches:

  • Screen natural product libraries against fungal growth with secondary validation on TRM61

  • Implement tRNA modification-specific reporter systems for high-throughput screening

  • Develop whole-cell assays measuring translation fidelity as a proxy for TRM61 function

  • Use chemical genetic approaches to identify synergistic compound combinations

• Selectivity Strategies:

  • Focus on unique structural features of the fungal enzyme compared to human homologs

  • Target fungal-specific regulatory mechanisms controlling TRM61 activity

  • Exploit differences in cellular localization or complex formation between fungal and human enzymes

  • Design compounds with preferential uptake by fungal cells

• Compound Optimization Workflow:

  Development Phase	Key Activities	Success Metrics
  Initial Screening	High-throughput biochemical assays	IC₅₀ < 10 μM against purified enzyme
  Hit Validation	Cellular activity, selectivity testing	MIC < 32 μg/mL; >10x selectivity vs. human enzyme
  Lead Optimization	SAR studies, ADME improvement	MIC < 4 μg/mL; acceptable toxicity profile
  Preclinical Testing	Animal models of fungal infection	Efficacy in invasive aspergillosis models
  Formulation	Develop appropriate delivery systems	Suitable for both topical and systemic administration

• Combination Therapy Potential:

  • Identify synergistic effects with established antifungals

  • Develop dual-targeting molecules affecting both TRM61 and other essential processes

  • Explore sequential therapy to prevent resistance development

  • Target TRM61 to sensitize resistant strains to conventional antifungals

• Biomarker Development:

  • Establish methods to measure tRNA m1A58 levels in clinical samples

  • Develop assays to monitor target engagement in vivo

  • Identify downstream metabolic signatures of successful TRM61 inhibition

  • Create tools to predict and monitor potential resistance mechanisms

The development of TRM61 inhibitors represents a promising approach for new antifungal therapeutics, particularly given the enzyme's essential role in fungal viability and the structural differences that could be exploited for selectivity against human homologs. The chronic and treatment-refractory nature of many invasive Aspergillus infections underscores the need for such novel therapeutic approaches.