Recombinant Neosartorya fumigata Diphthamide biosynthesis protein 4 (dph4)

What is the fundamental role of Dph4 in diphthamide biosynthesis?

Dph4 functions as a co-chaperone involved in diphthamide biosynthesis, particularly in the assembly of iron-sulfur clusters on Dph1 and Dph2. Unlike other Dph proteins, Dph4 does not co-localize with Dph1 and Dph2, suggesting it plays a distinct role in the biosynthetic pathway . The protein contains an N-terminal J domain commonly found in co-chaperones of 70 kilodalton heat shock proteins (Hsp70) and a C-terminal CSL zinc finger domain that preferentially binds iron over zinc . This dual domain structure enables Dph4 to potentially function in both electron transfer and co-chaperone activities, making it essential for the proper formation of diphthamide on elongation factor 2.

How does the structure of Dph4 compare across fungal species versus yeast and human orthologs?

While specific structural comparisons of Neosartorya fumigata Dph4 are not detailed in the available data, research on yeast Dph4 reveals a characteristic domain organization with an N-terminal J domain and a C-terminal CSL zinc finger . The J domain is functionally important and can be substituted with J domains from other proteins (such as Ydj1 in yeast) while maintaining activity . The CSL zinc finger binds iron more tightly than zinc, which induces protein oligomerization and confers redox activity . Comparative analysis across species would typically involve sequence alignment and structural prediction to identify conserved residues critical for function, particularly focusing on the J domain and CSL zinc finger regions.

What is the relationship between Dph4 and the EF2 modification pathway?

Dph4 is one of several proteins required for the multi-step pathway of diphthamide biosynthesis on EF2. In this pathway, Dph4 likely functions as a co-chaperone involved in the assembly of iron-sulfur clusters on Dph1 and Dph2 . These iron-sulfur clusters are critical for the first step of diphthamide biosynthesis, which involves the formation of a C-C bond between the target histidine residue in EF2 and the 3-amino-3-carboxypropyl group of S-adenosylmethionine (SAM) . Dph4 does not directly modify EF2 but instead enables other proteins in the pathway to function properly through its co-chaperone and potential electron transfer activities.

What are the optimal expression systems for producing recombinant N. fumigata Dph4?

• Selection of appropriate E. coli strains (BL21(DE3), Rosetta, or SHuffle for disulfide bond formation)

• Optimization of induction conditions (temperature: 16-25°C, IPTG concentration: 0.1-0.5 mM)

• Supplementation with iron (ferrous ammonium sulfate, 100-200 μM) during expression

• Growth under microaerobic conditions to preserve iron-sulfur cluster formation

• Addition of reducing agents (β-mercaptoethanol or DTT) to prevent oxidation

For structural studies requiring proper folding and metal incorporation, eukaryotic expression systems such as Pichia pastoris or insect cells may provide advantages for preserving the functional domains, particularly the J domain and CSL zinc finger region.

What purification challenges are specific to Dph4 and how can they be addressed?

Purification of Dph4 presents several challenges due to its iron-binding properties and domain structure. A comprehensive purification protocol should include:

• Anaerobic purification techniques to preserve iron-sulfur clusters

• Buffer optimization containing:

  • HEPES or Tris buffer (pH 7.5-8.0)

  • NaCl (150-300 mM)

  • Glycerol (10-15%)

  • Reducing agents (DTT or TCEP, 1-5 mM)

  • Protease inhibitors

• Multi-step purification approach:

  • Immobilized metal affinity chromatography (IMAC) using His-tag

  • Ion exchange chromatography to separate oligomeric states

  • Size exclusion chromatography for final polishing

• Spectroscopic verification of iron content using:

  • UV-Visible spectroscopy (characteristic absorption at ~400 nm)

  • Electron paramagnetic resonance (EPR) to confirm iron-sulfur cluster integrity

  • Inductively coupled plasma mass spectrometry (ICP-MS) for quantitative metal analysis

The CSL zinc finger domain's preferential binding of iron over zinc necessitates careful attention to metal content throughout purification .

How can the co-chaperone activity of N. fumigata Dph4 be effectively measured?

Based on the J domain function of Dph4 in other organisms , measuring co-chaperone activity of N. fumigata Dph4 would involve:

• ATPase stimulation assay:

  • Purification of partner Hsp70 chaperones

  • Measurement of ATP hydrolysis rates using malachite green phosphate assay

  • Calculation of stimulation factors in the presence of varying Dph4 concentrations

• Protein-protein interaction studies:

  • Pull-down assays with potential Hsp70 partners

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry to characterize thermodynamic parameters

• Client protein folding assays:

  • Using model substrates such as luciferase or citrate synthase

  • Measuring prevention of aggregation through light scattering

  • Assessing refolding efficiency after denaturation

The functional integrity of the J domain can be verified by comparing wild-type Dph4 with mutants containing alterations in the conserved HPD motif typically found in J domains.

What assays can demonstrate the iron-binding and redox properties of Dph4?

To characterize the iron-binding and redox properties associated with the CSL zinc finger domain of Dph4 , researchers can employ:

• Metal binding assays:

  • Competitive binding assays using specific chelators

  • Isothermal titration calorimetry to determine binding affinities

  • Inductively coupled plasma mass spectrometry (ICP-MS) for quantitative analysis

• Redox activity measurements:

  • Cyclic voltammetry to determine redox potentials

  • Oxygen consumption assays to measure electron transfer capabilities

  • NADH/NADPH oxidation assays as indicators of electron transfer activity

• Spectroscopic characterization:

  • UV-Visible spectroscopy to monitor characteristic absorption changes

  • Electron paramagnetic resonance (EPR) to identify redox states

  • Mössbauer spectroscopy for detailed iron electronic structure analysis

How can researchers reconstitute the diphthamide biosynthesis pathway in vitro to study Dph4 function?

An in vitro reconstitution approach to study Dph4 function within the diphthamide biosynthesis pathway would require:

• Component preparation:

  • Purification of all pathway proteins (Dph1-Dph7)

  • Preparation of substrate EF2 protein

  • SAM and other cofactors (ATP, GTP, Fe-S cluster precursors)

• Stepwise assembly and analysis:

  • Anaerobic reaction conditions with reducing agents

  • Incubation of EF2 with Dph1-Dph2 complex, SAM, and dithionite under anaerobic conditions as described for archaeal systems

  • Addition of purified Dph4 to assess enhancement of reaction efficiency

  • Mass spectrometric analysis to detect formation of ACP-modified EF2 intermediates

• Validation experiments:

  • Site-directed mutagenesis of key residues in both J domain and CSL zinc finger regions

  • Complementation assays using Dph4 variants in Dph4-deficient systems

  • Comparison of reaction rates and product formation with and without functional Dph4

This approach parallels the successful reconstitution of archaeal diphthamide biosynthesis described for Pyrococcus horikoshii , adapted to include the additional components required in eukaryotic systems.

Which amino acid residues in N. fumigata Dph4 are critical for its function in diphthamide biosynthesis?

Critical residues in Dph4 would include:

• J domain functional residues:

  • The HPD motif, which is essential for stimulating Hsp70 ATPase activity

  • Helix II and III residues that form the Hsp70 interaction surface

  • N-terminal region residues that determine specificity for particular Hsp70 partners

• CSL zinc finger domain residues:

  • Conserved cysteine residues critical for iron coordination

  • Hydrophobic core residues that stabilize the zinc finger fold

  • Surface residues involved in potential protein-protein interactions

• Interdomain linker residues:

  • Flexible regions that allow proper orientation of functional domains

  • Potential regulatory sites for post-translational modifications

A systematic mutagenesis approach coupled with functional assays would be required to precisely identify critical residues. Based on yeast studies, the conserved cysteine residue in the CSL sequence is particularly important for Dph4 activity .

How does the oligomerization state of Dph4 affect its function in diphthamide biosynthesis?

Iron binding to the CSL zinc finger domain of Dph4 induces oligomerization, which may be functionally significant . To investigate this relationship:

• Oligomerization analysis techniques:

  • Size exclusion chromatography to separate oligomeric states

  • Analytical ultracentrifugation to determine stoichiometry

  • Dynamic light scattering to assess homogeneity

  • Native PAGE to visualize discrete oligomeric species

• Structure-function correlation:

  • Activity assays with isolated monomeric versus oligomeric forms

  • Cross-linking experiments to stabilize specific oligomeric states

  • Electron microscopy to visualize oligomeric arrangements

• Iron-dependence studies:

  • Manipulation of iron:protein ratios to control oligomerization

  • Chelation experiments to determine reversibility

  • Mutagenesis of key residues involved in oligomerization interfaces

Understanding the relationship between oligomerization and function could provide insights into the regulation of diphthamide biosynthesis and potential approaches for modulating this activity.

How does N. fumigata Dph4 differ from homologs in other fungi and in higher eukaryotes?

A comprehensive comparative analysis would examine:

• Sequence conservation patterns:

  • Multiple sequence alignment of Dph4 proteins across species

  • Identification of highly conserved versus variable regions

  • Phylogenetic analysis to establish evolutionary relationships

• Domain architecture comparison:

  • Conservation of J domain and CSL zinc finger arrangements

  • Presence of additional domains or regulatory elements

  • Variations in linker regions between functional domains

• Functional equivalence testing:

  • Cross-species complementation assays

  • Domain swapping experiments between orthologs

  • Heterologous expression and functional testing

Notably, while yeast Dph4 contains both J domain and CSL zinc finger domains , the human ortholog may have functional specializations that could inform understanding of the fungal protein's role.

What are the evolutionary implications of Dph4's role in the diphthamide modification pathway?

The evolutionary aspects of Dph4 function include:

• Conservation across domains of life:

  • Presence of diphthamide biosynthesis machinery in eukaryotes and archaea

  • Absence of diphthamide in bacteria

  • Differences between archaeal and eukaryotic pathways (e.g., archaea use a homodimeric Dph2 while eukaryotes use a DPH1-DPH2 heterodimer)

• Functional adaptation:

  • Role specialization between different Dph proteins

  • Co-evolution with translation machinery

  • Selection pressures related to toxin resistance (e.g., diphtheria toxin targets diphthamide)

• Structural evolution:

  • Domain acquisition or loss across lineages

  • Adaptation of metal binding properties

  • Development of regulatory mechanisms

Interestingly, while archaeal diphthamide biosynthesis can be reconstituted with just Dph2 in vitro , eukaryotic systems require multiple proteins including Dph4, suggesting increased pathway complexity during evolution.

How can structure-guided mutagenesis of Dph4 provide insights into the mechanism of diphthamide biosynthesis?

Structure-guided mutagenesis approaches should include:

This approach can particularly illuminate how Dph4's co-chaperone activity contributes to the assembly of iron-sulfur clusters on Dph1 and Dph2, which are critical for the first step of diphthamide biosynthesis .

What methodologies are most effective for studying the interaction between Dph4 and other diphthamide biosynthesis proteins?

To investigate protein-protein interactions involving Dph4:

• In vitro interaction studies:

  • Pull-down assays using tagged Dph4 and other pathway components

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic characterization

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Cellular interaction validation:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Proximity ligation assay (PLA)

  • Co-immunoprecipitation from native systems

• Structural characterization:

  • Cryo-electron microscopy of complexes

  • X-ray crystallography of co-crystallized components

  • Cross-linking mass spectrometry to identify proximal residues

  • Integrative structural modeling combining multiple data sources

While Dph4 does not co-localize with Dph1 and Dph2 , it may have transient interactions or function through other intermediaries, requiring sensitive detection methods.

How can researchers develop assays to monitor the iron-sulfur cluster assembly function of Dph4 in real-time?

Real-time monitoring of Dph4-mediated iron-sulfur cluster assembly would involve:

• Spectroscopic approaches:

  • Continuous UV-visible spectroscopy to track characteristic iron-sulfur cluster absorption

  • Fluorescence-based sensors for iron-sulfur cluster formation

  • EPR spectroscopy with rapid-freeze quenching at different time points

  • Resonance Raman spectroscopy for cluster coordination environment

• Electrochemical methods:

  • Protein film voltammetry to monitor redox changes during assembly

  • Microelectrode measurements of electron transfer activity

  • Potentiometric titrations to determine redox transitions

• Structural techniques:

  • Time-resolved small-angle X-ray scattering to monitor conformational changes

  • Hydrogen-deuterium exchange mass spectrometry at defined time points

  • Stopped-flow X-ray absorption spectroscopy for metal coordination changes

These approaches would help elucidate the kinetics and mechanism of Dph4's co-chaperone activity in iron-sulfur cluster assembly on Dph1 and Dph2, particularly focusing on how the J domain and CSL zinc finger functions are coordinated during this process .