Recombinant Neurospora crassa Diphthamide biosynthesis protein 2 (dph-2), partial

What is the molecular role of Diphthamide biosynthesis protein 2 in Neurospora crassa?

Diphthamide biosynthesis protein 2 (dph-2) in Neurospora crassa is a crucial component of the diphthamide synthesis pathway, involved in the first step of modifying elongation factor 2 (EF2). The protein functions by transferring a 3-amino-3-carboxypropyl group from S-adenosylmethionine (SAM) to the histidine residue of EF2. Unlike in archaea where Dph2 functions as a homodimer, in eukaryotes like N. crassa, Dph1 and Dph2 form a heterodimer to catalyze this first step. The reaction involves a novel iron-sulfur cluster mechanism where the [4Fe-4S] cluster breaks the C-γ,Met-S bond of SAM and generates a 3-amino-3-carboxypropyl radical, unlike typical radical SAM enzymes that form a 5'-deoxyadenosyl radical .

How is the dph-2 gene regulated in Neurospora crassa compared to other fungi?

The regulation of dph-2 in Neurospora crassa follows a pattern distinct from that of Saccharomyces cerevisiae. While yeast requires both DPH1 and DPH2 genes for diphthamide biosynthesis, evidence indicates that regulation in Neurospora involves interactions with circadian clock elements. Recent studies suggest connections between casein kinase I (CKI) levels and dph-2 expression, as observed in period mutants in Neurospora. The PRD-2 protein affects CKI transcript stability, potentially influencing dph-2 expression indirectly through translation regulation mechanisms . Additionally, unlike S. cerevisiae where DPH3 and DPH4 are required for Fe-S cluster assembly and electron transfer, the regulatory network in Neurospora appears to be more tightly integrated with basic growth and developmental pathways .

What experimental methods are typically used to characterize dph-2 activity in filamentous fungi?

Characterization of dph-2 activity in filamentous fungi like Neurospora crassa typically employs multiple complementary approaches:

• Genetic approaches: Gene knockouts using homologous recombination or CRISPR-Cas9 systems, followed by phenotypic analysis of growth rates and morphology .

• Protein purification: Recombinant expression in E. coli, yeast, or mammalian cell systems, followed by purification using affinity chromatography methods such as hydrophobic interaction chromatography (HIC) with PHE-Sepharose and ion exchange chromatography with Q15-Source .

• Activity assays: In vitro reconstruction of diphthamide synthesis using purified components and detection of product formation using techniques like LC-MS or NMR spectroscopy .

• ADP-ribosylation assays: Using diphtheria toxin to assess the presence of functional diphthamide on EF2 .

• Iron-sulfur cluster characterization: UV-Vis, EPR, and Mössbauer spectroscopies to analyze the [4Fe-4S] cluster essential for Dph2 function .

How can recombinant Neurospora crassa dph-2 be effectively expressed and stabilized for structural studies?

Effective expression and stabilization of recombinant Neurospora crassa dph-2 for structural studies requires careful consideration of the protein's oxygen-sensitive [4Fe-4S] cluster. A recommended protocol involves:

• Expression system optimization: Use of Pichia pastoris with the methanol-inducible AOX1 promoter system has proven effective. Transformation with linearized plasmids containing the dph-2 gene under AOX1 control yields better results than E. coli expression systems .

• Anaerobic purification: All purification steps must be conducted under strict anaerobic conditions to preserve the [4Fe-4S] cluster. This typically requires a glove box filled with nitrogen and specialized equipment .

• Buffer composition: Inclusion of reducing agents like dithionite (5-10 mM) and DTT (1-5 mM) in all buffers is essential. A typical buffer composition includes 50 mM phosphate buffer (pH 5.5-6.5), 137 mM NaCl, and 10% glycerol .

• Iron-sulfur cluster reconstitution: After initial purification, reconstitution of the [4Fe-4S] cluster using ferrous ammonium sulfate, sodium sulfide, and DTT under anaerobic conditions significantly improves protein stability and activity .

• Storage considerations: Addition of 50% glycerol and storage at -80°C in sealed, oxygen-free containers helps maintain protein integrity. The shelf life of lyophilized protein is approximately 12 months at -20°C/-80°C, while liquid preparations typically remain stable for 6 months .

What are the methodological challenges in assessing the interaction between dph-2 and other diphthamide biosynthesis proteins in Neurospora?

Methodological challenges in studying dph-2 interactions with other diphthamide biosynthesis proteins in Neurospora include:

• Oxygen sensitivity: The [4Fe-4S] cluster in dph-2 is extremely oxygen-sensitive, making traditional protein-protein interaction studies like co-immunoprecipitation technically challenging. Solutions include performing all experiments in anaerobic chambers with specialized equipment .

• Transient interactions: The interactions between diphthamide biosynthesis proteins appear to be transient and potentially dependent on the presence of EF2, making them difficult to capture. Crosslinking approaches with carefully optimized conditions are necessary to detect these interactions .

• Functional redundancy: In eukaryotes, functional redundancy between some diphthamide biosynthesis proteins complicates interpretation of results. For example, distinguishing the specific contributions of dph-1 versus dph-2 when they form a heterodimer .

• Native expression levels: The relatively low natural expression levels of diphthamide biosynthesis proteins necessitate sensitive detection methods. Developing specific antibodies against N. crassa dph proteins has proven challenging .

• Complex formation dynamics: Evidence suggests that dissociation of dph-5 from EF2 is required for dph-6 to perform the final amidation step, indicating the need to develop methods that can capture the dynamic assembly and disassembly of these protein complexes .

How does the recombinant N. crassa dph-2 differ functionally from its homologs in archaea and other eukaryotes?

The functional differences between recombinant N. crassa dph-2 and its homologs in archaea and other eukaryotes are significant:

• Structural organization: While archaeal Dph2 (e.g., from P. horikoshii) functions as a homodimer with each monomer containing a [4Fe-4S] cluster, N. crassa dph-2 (like other eukaryotic homologs) forms a heterodimer with dph-1. The eukaryotic dph-1 retains all three conserved cysteine residues for [4Fe-4S] cluster binding, while dph-2 has only two of these residues .

• Iron-sulfur cluster coordination: The partial conservation of cysteine residues in N. crassa dph-2 suggests that the heterodimer of dph-1-dph-2 likely binds only one [4Fe-4S] cluster, primarily through dph-1, compared to two clusters in the archaeal homodimer .

• Dependency on additional factors: Unlike archaeal systems where Dph2 alone is sufficient for the first step of diphthamide biosynthesis in vitro, the N. crassa pathway requires additional proteins (dph-3 and dph-4), which may function in maintaining the [4Fe-4S] cluster in a reduced state or assisting in its assembly .

• Cellular localization: Studies suggest that N. crassa dph-2 may be predominantly cytoplasmic, while some evidence indicates that the human homolog may also have nuclear functions .

What is the recommended protocol for expressing and purifying functional recombinant N. crassa dph-2?

The recommended protocol for expressing and purifying functional recombinant N. crassa dph-2 consists of several critical steps:

Expression system and growth conditions:

• Clone the dph-2 gene into pPICZα A vector under the control of the methanol-inducible AOX1 promoter

• Transform into electrocompetent P. pastoris cells

• Select transformants on YPDS plates containing zeocin (100 μg/ml)

• Pre-culture in BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% yeast nitrogen base, 0.4 mg/L biotin, 1% glycerol) at 30°C until OD600 reaches 2-6

• Induce expression by transferring to BMMY medium containing 0.5-1% methanol

• Maintain induction by adding methanol (0.5% final concentration) every 24 hours for 3-4 days

• Harvest cells by centrifugation at 5,000 × g for 15 minutes at 4°C

Purification procedure:

• Resuspend cells in lysis buffer (50 mM sodium phosphate pH 6.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, 5 mM DTT)

• Lyse cells using glass beads in a bead beater (10 cycles of 30 seconds on/30 seconds off)

• Clarify lysate by centrifugation at 20,000 × g for 20 minutes at 4°C

• Load supernatant onto a PHE-Sepharose Fast Flow column equilibrated with 50 mM phosphate buffer (pH 5.5) containing 20% ammonium sulfate

• Elute within a linear gradient from 20 to 0% ammonium sulfate within 5 column volumes

• Pool active fractions and dialyze against 50 mM sodium acetate buffer (pH 5.5)

• Apply to a Q15-Source column and elute with a linear salt gradient from 0 to 1 M NaCl

• For highest purity, perform a final gel filtration step using Superdex 200

Iron-sulfur cluster reconstitution:

• All subsequent steps must be performed anaerobically in a glove box (≤1 ppm O2)

• Add 10 molar equivalents of FeCl3 and Na2S, and 5 mM DTT to purified protein

• Incubate overnight at 4°C

• Remove excess iron and sulfide by gel filtration chromatography

• Verify cluster formation by UV-visible spectroscopy (characteristic absorption at ~410 nm)

How can researchers accurately measure the enzymatic activity of recombinant dph-2 in vitro?

Accurate measurement of recombinant dph-2 enzymatic activity in vitro requires specialized methods designed to detect the unusual radical SAM chemistry involved:

SAM cleavage assay:

• Prepare reaction mixture containing: purified dph-2 (1-5 μM), [14C]-SAM (50-100 μM), sodium dithionite (1-2 mM), DTT (5 mM), and buffer (50 mM HEPES pH 7.5, 150 mM NaCl)

• Incubate anaerobically at 30°C for 30-60 minutes

• Analyze SAM cleavage products by TLC and quantify radioactivity in 5'-methylthioadenosine (MTA) spot

Complete diphthamide synthesis assay:

• Include purified EF2 (1-5 μM) in the reaction mixture above

• After incubation, precipitate protein with trichloroacetic acid

• Resuspend precipitate in buffer and analyze by MALDI-MS to detect the mass shift corresponding to ACP addition to the target histidine residue

ADP-ribosylation assay:

• After the in vitro diphthamide synthesis reaction, add diphtheria toxin (100 ng) and [32P]-NAD+ (5 μM)

• Incubate at 37°C for 30 minutes

• Precipitate proteins with TCA, wash, and quantify radioactivity incorporation

• Alternatively, perform western blot analysis using antibodies specific to ADP-ribosylated EF2

Iron-sulfur cluster spectroscopic analysis:

• Monitor [4Fe-4S] cluster integrity by UV-visible spectroscopy (characteristic absorption at ~410 nm)

• For detailed characterization, perform electron paramagnetic resonance (EPR) spectroscopy after reduction with dithionite to detect the [4Fe-4S]+ state

• Mössbauer spectroscopy can provide definitive evidence for cluster type and oxidation state

What are the key considerations when designing experiments to investigate the interaction between N. crassa dph-2 and elongation factor 2?

When designing experiments to investigate interactions between N. crassa dph-2 and elongation factor 2, researchers should consider:

Protein preparation considerations:

• Anaerobic conditions: All experiments must be conducted under strict anaerobic conditions (preferably in a glove box with ≤1 ppm O2) to prevent degradation of the oxygen-sensitive [4Fe-4S] cluster in dph-2 .

• Protein tagging strategy: N-terminal tags are preferred over C-terminal tags for dph-2, as C-terminal modifications may interfere with substrate recognition. For EF2, avoid tagging near His699 (the diphthamide modification site) .

• Buffer optimization: Use buffers that maintain [4Fe-4S] cluster integrity (typically including 5 mM DTT) while supporting EF2 stability. HEPES buffer (50 mM, pH 7.5) with 150 mM NaCl and 10% glycerol is often effective .

Interaction detection methods:

• Co-immunoprecipitation: Perform under anaerobic conditions using antibodies against dph-2 or EF2. Include controls with alanine substitution at His699 in EF2 to verify specific interaction at the modification site .

• Crosslinking approaches: Use mild crosslinkers like formaldehyde (0.1-1%) or DSS (disuccinimidyl suberate) to capture transient interactions. Optimize crosslinking time carefully (typically 5-30 minutes) .

• Biolayer interferometry/SPR: For kinetic analysis of binding, immobilize one protein (preferably EF2) and measure association/dissociation of dph-2. Ensure that immobilization does not interfere with the interaction site .

Functional validation strategies:

• In vitro reconstitution: Combine purified dph-2, dph-1, EF2, and SAM under anaerobic conditions, then analyze by mass spectrometry to detect successful transfer of the ACP group to EF2 .

• Diphtheria toxin sensitivity: After in vitro reaction, test the modified EF2 for increased sensitivity to ADP-ribosylation by diphtheria toxin compared to unmodified EF2 .

• Mutational analysis: Create point mutations in conserved residues of dph-2, particularly the cysteines involved in [4Fe-4S] cluster coordination, and assess effects on EF2 binding and modification .

How should researchers interpret contradictory results when analyzing dph-2 function in different experimental systems?

When encountering contradictory results regarding dph-2 function across different experimental systems, researchers should consider several factors for proper interpretation:

• Iron-sulfur cluster integrity: The [4Fe-4S] cluster in dph-2 is highly oxygen-sensitive. Verify cluster integrity in each experimental system using UV-visible spectroscopy (characteristic absorption at ~410 nm). Loss of cluster activity can occur without visible protein degradation, leading to apparently contradictory results .

• Heterodimer formation: Unlike archaeal systems where Dph2 functions as a homodimer, eukaryotic dph-2 requires dph-1 to form a functional heterodimer. Confirm whether your experimental system contains both proteins in the appropriate stoichiometry .

• Species-specific differences: Compare sequence alignments across species. Key differences exist between archaeal and eukaryotic systems, and even between fungal species like S. cerevisiae and N. crassa. For example, in DPH2 variants, mutations at position H123P in yeast show different severity of functional impairment compared to the corresponding H105P in human cells .

• Dependency on additional factors: While archaeal Dph2 alone can catalyze the first step of diphthamide synthesis in vitro, eukaryotic systems require additional proteins (dph-3 and dph-4). Verify the presence of these cofactors in your experimental system .

• Influence of experimental conditions: Document and compare precise buffer compositions, pH, salt concentrations, and reducing agents across experiments. Small variations can significantly impact dph-2 activity. For instance, dithionite at 1-2 mM concentration is often essential for in vitro activity .

What are common technical challenges when working with recombinant N. crassa dph-2 and how can they be addressed?

Common technical challenges when working with recombinant N. crassa dph-2 and their solutions include:

How can researchers distinguish between specific effects of dph-2 mutation and downstream consequences in phenotypic analyses?

Distinguishing between direct effects of dph-2 mutation and downstream consequences requires systematic approaches:

• Complementation studies: Reintroduce wild-type dph-2 into mutant strains to verify phenotype rescue. Include controls with catalytically inactive dph-2 variants (e.g., mutations in iron-sulfur cluster coordination sites) to distinguish between structural and catalytic roles .

• Temporal analysis: Monitor phenotypic changes over time following controlled induction or repression of dph-2. This helps identify primary effects (occurring rapidly) versus secondary consequences (developing gradually) .

• Differential gene expression analysis: Compare transcriptome profiles between wild-type and dph-2 mutant strains under identical conditions. Direct effects of dph-2 mutation should affect a specific subset of genes, while widespread changes suggest secondary consequences .

• Biochemical validation: Directly measure diphthamide modification levels on EF2 using mass spectrometry or diphtheria toxin ADP-ribosylation assays. This connects genotype to molecular phenotype before examining cellular consequences .

• Domain-specific mutations: Create targeted mutations in different functional domains of dph-2 to separate roles. For example, mutations in the [4Fe-4S] cluster coordination sites versus potential protein-protein interaction interfaces .

• Cross-species comparison: Compare phenotypes of dph-2 mutations across different fungal species where the diphthamide pathway is conserved. Consistent phenotypes across species likely represent direct effects .

• Genetic interaction mapping: Perform systematic genetic interaction screens to identify genes whose mutation exacerbates or suppresses dph-2 mutant phenotypes. This helps place dph-2 in functional pathways and distinguish direct from indirect effects .

How might dph-2 function intersect with other cellular pathways in Neurospora crassa?

Recent evidence suggests significant intersections between dph-2 function and several cellular pathways in Neurospora crassa:

• Circadian rhythm regulation: Studies of period mutants in N. crassa have revealed connections between diphthamide biosynthesis and circadian clock regulation. The PRD-2 protein, an RNA-binding protein that affects clock period length, regulates casein kinase I transcript stability, which may indirectly influence dph-2 expression through translation regulation mechanisms .

• Stress response pathways: N. crassa responds to oxidative and other stresses by activating alternative respiration pathways. The two zinc-cluster transcription factors that control induction of alternative oxidase (AOD-2 and AOD-5) may also influence diphthamide biosynthesis genes under stress conditions, suggesting coordination between energy metabolism and translation accuracy .

• Cell membrane biophysics: Changes in cell membrane properties during growth and development in N. crassa correlate with alterations in translation patterns. The diphthamide modification on EF2 may play a role in optimizing translation under different membrane environments, as suggested by fluorescence spectroscopy studies on N. crassa membrane properties during development .

• Heterochromatin formation: Recent discoveries about H3T11 phosphorylation by casein kinase II (CKII) and its requirement for heterochromatin formation in N. crassa suggest potential connections with diphthamide biosynthesis. Both pathways involve precise regulation of fundamental cellular processes, and preliminary evidence suggests they may be co-regulated under certain conditions .

• Amino acid and purine metabolism: Diphthamide biosynthesis requires SAM as a key substrate, connecting this pathway to one-carbon metabolism. In N. crassa, mutations in histidine biosynthesis genes (e.g., his-3) have shown synthetic phenotypes with diphthamide pathway mutations, suggesting metabolic crosstalk .

What are the most promising approaches for studying the structural biology of N. crassa dph-2 in complex with its partners?

The most promising approaches for structural studies of N. crassa dph-2 in complex with its partners include:

• Cryo-electron microscopy (cryo-EM): Given the large size of the dph-1/dph-2 heterodimer (~120 kDa) and its interaction with EF2 (~95 kDa), cryo-EM represents the most promising approach. Sample preparation must be performed anaerobically to preserve the [4Fe-4S] cluster. Single-particle analysis can potentially achieve 3-4 Å resolution, sufficient to visualize the interaction interface and the position of the [4Fe-4S] cluster .

• Integrated structural biology approaches: Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS) with small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry (XL-MS) can provide complementary structural information while requiring less protein than crystallography. These methods are particularly valuable for capturing the dynamic nature of dph-2 interactions .

• NMR spectroscopy of isolated domains: While full-length dph-2 is too large for conventional NMR, individual domains (~20-30 kDa) can be studied by solution NMR. This approach is particularly useful for examining how specific domains interact with substrates or binding partners .

• Computational modeling with experimental validation: Using the archaeal Dph2 crystal structure as a template, homology models of N. crassa dph-2 can be generated and refined using molecular dynamics simulations. These models can then be validated through site-directed mutagenesis and functional assays .

• Protein engineering approaches: Creation of stabilized variants through surface entropy reduction (SER) or the introduction of disulfide bonds may enhance the likelihood of successful crystallization. Additionally, antibody-mediated crystallization using Fab fragments may facilitate crystal packing while allowing capture of specific conformational states .

What are the implications of understanding N. crassa dph-2 for applications in biotechnology and medicine?

Understanding N. crassa dph-2 has several significant implications for biotechnology and medicine:

• Novel antimicrobial development: The diphthamide biosynthesis pathway is conserved in fungi but absent in bacteria, making it a potential target for antifungal development. Selective inhibitors of fungal dph-2 could lead to translation disruption specifically in fungal pathogens. N. crassa serves as an excellent model system for such drug development due to its genetic tractability .

• Protection against bacterial toxins: Understanding how diphthamide biosynthesis proteins function could lead to strategies for protecting cells against diphtheria toxin and Pseudomonas exotoxin A, which target diphthamide-modified EF2. Structural insights from N. crassa dph-2 could inform the design of molecules that protect the diphthamide residue without disrupting EF2 function .

• Understanding genetic disorders: Mutations in human DPH genes are associated with developmental disorders and intellectual disability. The dph-2 mutation H105P in human cells shows significantly reduced diphthamide synthase activity compared to wild-type. Detailed mechanistic understanding of dph-2 from model organisms like N. crassa provides insights into how these mutations affect human health .

• Recombinant protein production: The translation accuracy conferred by diphthamide-modified EF2 is particularly important for the production of complex recombinant proteins. Optimizing diphthamide biosynthesis in expression hosts could enhance the fidelity of protein synthesis, potentially improving yields of difficult-to-express proteins .

• Biomarker development: The diphthamide modification status of EF2 could serve as a biomarker for certain cellular states or diseases. Antibodies or other detection methods specifically targeting this modification could be developed based on structural insights from studies of the N. crassa pathway .

• Understanding neurodegenerative connections: Recent evidence links diphthamide deficiency to neurological disorders. For example, studies show an association between Darier disease (caused by SERCA2 dysfunction) and neurodegenerative disorders. Since the diphthamide pathway involves iron-sulfur clusters similar to those in other neurodegenerative disease-associated proteins, mechanistic parallels may exist .