Recombinant Ustilago maydis NADH-ubiquinone oxidoreductase chain 4L (ND4L)

How does Ustilago maydis ND4L structure compare to homologous proteins in other species?

The ND4L protein in Ustilago maydis shares significant structural similarities with homologous proteins from other species, particularly in the transmembrane regions involved in proton translocation. While specific structural data for U. maydis ND4L is limited, homology modeling approaches similar to those used for human ND4L can be applied. The protein typically contains multiple transmembrane helices that create channels for proton movement across the inner mitochondrial membrane. Key conserved residues, such as those forming the proton translocation pathway (including glutamate residues), are generally preserved across species. Molecular dynamics simulations of ND4L-ND6 subunit interactions reveal characteristic water channels and hydrogen bond networks that are essential for proton movement . These structural features are likely conserved in Ustilago maydis, allowing for comparative structural analyses with homologous proteins from model organisms.

What are the key amino acid residues involved in proton translocation in ND4L?

The proton translocation function of ND4L depends on several conserved amino acid residues that form the transmembrane channel. Molecular dynamics simulations have identified glutamate residues, particularly Glu34, as critical for the proton translocation pathway. These negatively charged residues form part of a hydrogen bond network that facilitates proton movement across the membrane. Additionally, interactions with tyrosine residues (such as Tyr157) have been observed to be important in maintaining the proper configuration of the proton channel .

In functional studies, mutations affecting these key residues significantly disrupt proton translocation. For example, molecular dynamics simulations of mutations like M47T and C69W demonstrate how single amino acid changes can interrupt the translocation pathway by altering hydrogen bond formations between Glu34 and Tyr157, restricting the passage of water molecules through the transmembrane region . This suggests that these conserved residues are essential for maintaining the proper structure and function of the proton channel in ND4L.

What are the optimal expression systems for producing recombinant Ustilago maydis ND4L?

Expression of recombinant Ustilago maydis ND4L presents challenges due to its hydrophobic nature and involvement in multi-subunit complex formation. Based on similar membrane protein studies, the following expression systems have shown promise:

For functional studies, co-expression with other complex I components may be necessary to achieve proper folding and assembly. When expressing ND4L independently, fusion partners such as GFP or MBP have been shown to improve solubility and membrane localization. The choice of detergent during purification is critical, with mild detergents like DDM (n-dodecyl-β-D-maltoside) helping maintain protein structure . Successful expression has been achieved by designing backbone proteins that ensure proper localization to the inner membrane, similar to the approach used for the NuoK+PufL fusion protein system .

How can researchers assess the proton translocation activity of recombinant ND4L in vitro?

Assessing proton translocation activity of recombinant ND4L requires specialized techniques that measure changes in membrane potential and proton gradients. A comprehensive methodology includes:

• Liposome Reconstitution Assay:

  • Purified ND4L is reconstituted into liposomes with defined lipid composition

  • Proton translocation is measured using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine)

  • The assay can quantify proton flux in response to electron transfer substrates

• Membrane Potential Measurements:

  • Similar to techniques applied for biogenic photosystems, potential-sensitive dyes like JC-1 or DiSC3(5) can monitor changes in membrane potential

  • This approach enables real-time analysis of proton-motive force generation

• Surface Photovoltage (SPV) Technique:

  • This method, adapted from photosystem analysis, can detect electron movement across the membrane

  • SPV measurements can be particularly useful when coupling ND4L activity to light-activated systems

• Coupled Enzymatic Assays:

  • NADH oxidation can be monitored spectrophotometrically at 340 nm

  • Ubiquinone reduction can be tracked at 275 nm

  • The ratio between these activities provides insights into coupling efficiency

What mutagenesis approaches are most effective for studying ND4L function?

Investigating ND4L function through mutagenesis requires strategic approaches that address the challenges of membrane protein manipulation. The most effective strategies include:

• Site-Directed Mutagenesis:

  • Target conserved residues identified through sequence alignment and homology modeling

  • Focus on amino acids like glutamate residues involved in proton channels

  • Create systematic alanine scanning libraries of transmembrane segments

  • Investigate naturally occurring mutations like those corresponding to 10609T>C (M47T) and 10663T>C (V65A) identified in human studies

• Domain Swapping:

  • Exchange transmembrane domains between ND4L from different species to identify functional regions

  • Create chimeric proteins to map species-specific functional differences

• In vivo Assessment Systems:

  • Utilize complementation assays in U. maydis or yeast complex I mutants

  • Measure respiratory capacity through oxygen consumption assays

  • Assess growth under various carbon sources to determine functional impairment

When designing mutagenesis experiments, researchers should consider combining computational prediction (molecular dynamics simulation) with experimental validation. For example, mutations that show disruption of proton pathways in silico can be prioritized for in vitro and in vivo testing. This integrated approach has successfully identified critical residues in ND4L such as those forming hydrogen bonds that maintain the proton translocation channel integrity .

How does ND4L interact with other components of the respiratory chain in Ustilago maydis?

The interaction between ND4L and other respiratory chain components in Ustilago maydis represents a complex network essential for energy metabolism. ND4L functions within the membrane arm of complex I, interacting with several other subunits to form a functional proton translocation pathway. Specifically:

• Intra-Complex I Interactions:

  • ND4L forms close associations with ND6 subunits, creating a functional module involved in proton translocation

  • This interaction creates water channels across the membrane that facilitate proton movement

  • Hydrophobic interactions between transmembrane helices stabilize these associations

• Quinone Binding Site Proximity:

  • Although not directly involved in quinone binding, ND4L's position influences electron transfer to the quinone pool

  • The quinone reduction pathway in U. maydis shows non-additive behavior with other respiratory components, suggesting competition for the quinone/quinol pool

• Alternative Respiratory Components:

  • U. maydis contains an external NADH dehydrogenase (NDH-2) that contributes equally to complex I in NADH-dependent respiratory activity

  • Unlike plant NDH-2, the U. maydis enzyme is not modulated by Ca²⁺

  • ND4L function must be understood in the context of this parallel electron input system

• Supercomplexes Formation:

  • In many organisms, complex I forms supercomplexes with complexes III and IV

  • These associations enhance electron transfer efficiency and reduce reactive oxygen species generation

  • Research suggests that specific ND4L residues may contribute to these larger assemblies

Understanding these interactions requires advanced techniques such as cryo-electron microscopy, crosslinking studies, and blue native gel electrophoresis to capture the native protein associations intact.

What role does ND4L play in cellular response to oxidative stress in Ustilago maydis?

ND4L's role in oxidative stress response in Ustilago maydis is multifaceted, stemming from its position in the respiratory chain and potential involvement in reactive oxygen species (ROS) generation. Current research indicates:

• ROS Production and Regulation:

  • As part of complex I, ND4L contributes to sites of electron leakage that can generate superoxide

  • Dysfunction in ND4L can increase ROS production, triggering oxidative stress

  • U. maydis contains alternative oxidase (AOX) that serves as an "overflow valve" for electrons, reducing ROS generation

• Adaptive Responses:

  • Under conditions of respiratory chain inhibition (e.g., antimycin A or cyanide treatment), the AOX pathway increases to approximately 75% of the uninhibited respiratory rate

  • This suggests a compensatory mechanism when conventional electron transport is compromised

  • ND4L mutations may activate similar stress response pathways

• Metabolic Rewiring:

  • Impaired ND4L function may necessitate metabolic adaptations including:

    • Increased glycolytic flux to compensate for ATP production

    • Altered NAD⁺/NADH ratios affecting numerous cellular processes

    • Upregulation of alternative NADH oxidation systems

• Potential Therapeutic Applications:

  • Understanding ND4L's role in oxidative stress provides insights for addressing mitochondrial disorders

  • Compounds targeting alternative respiratory pathways may offer protective effects when ND4L function is compromised

Research methodologies include measuring ROS levels using fluorescent probes, assessing mitochondrial membrane potential, and analyzing expression of stress response genes following manipulation of ND4L expression or activity.

How can molecular dynamics simulations guide experimental design for ND4L structure-function studies?

Molecular dynamics (MD) simulations provide powerful insights for experimental design in ND4L research, offering atomic-level details of protein behavior that can guide wet-lab investigations:

• Identification of Critical Residues:

  • MD simulations can reveal key amino acids involved in proton translocation

  • For example, simulations of ND4L-ND6 subunits have identified specific hydrogen bond networks essential for proton movement

  • Mutations like M47T and C69W have been shown through MD to disrupt proton pathways by forming new hydrogen bonds between Glu34 and Tyr157, providing specific targets for mutagenesis

• Water Channel Visualization:

  • Simulations track water molecule movement through the transmembrane region

  • This identifies the precise path of proton translocation and potential bottlenecks

  • Experimental validation can then target these regions through cysteine scanning or proton flux measurements

• Conformational Dynamics Analysis:

  • MD reveals protein motion over time, identifying flexible regions and rigid domains

  • This information helps design more stable recombinant constructs with preserved functional regions

  • Conformational changes in response to membrane potential can be predicted and tested experimentally

• Mutation Impact Prediction:

  • Prior to laboratory work, simulations can screen potential mutations for functional impact

  • This allows prioritization of experimental resources toward mutations with highest predicted effect

  • The table below summarizes simulation-predicted impacts of key mutations:

By combining 100ns or longer MD simulations with experimental validation, researchers can develop more targeted approaches to understanding ND4L function . This integrated strategy has successfully predicted functional impacts of mutations associated with human mitochondrial disorders, suggesting similar approaches would be valuable for Ustilago maydis ND4L research.

How can Ustilago maydis ND4L serve as a model for human mitochondrial diseases?

Ustilago maydis ND4L provides a valuable model system for studying human mitochondrial diseases, particularly those involving complex I dysfunction. The strategic advantages of this model include:

• Structural Conservation:

  • Despite evolutionary distance, the core functional domains and critical residues of ND4L are conserved between fungi and humans

  • Molecular modeling approaches can leverage this conservation for structural comparisons

  • Mutations identified in human patients can be recreated in corresponding positions in U. maydis ND4L

• Disease-Relevant Mutations:

  • Human mutations such as T10663C (Val65Ala) in MT-ND4L associated with Leber hereditary optic neuropathy (LHON) can be studied in the fungal system

  • The concurrent mutations observed in human LHON patients (10609T>C and 10663T>C) can be introduced to study cumulative effects

  • U. maydis provides a simpler genetic background for isolating mutation effects

• Functional Assay Advantages:

  • U. maydis grows rapidly and is genetically tractable

  • The presence of both conventional and alternative respiratory pathways allows assessment of compensatory mechanisms

  • Phenotypic readouts such as growth rate and oxygen consumption provide quantitative measures of mitochondrial function

• Translational Research Applications:

  • Screening potential therapeutic compounds in the U. maydis system before advancing to mammalian models

  • Identifying genetic suppressors that could represent targets for therapeutic intervention

  • Developing biomarkers for mitochondrial dysfunction

The connection between ND4L mutations and human diseases extends beyond LHON. Recent studies have investigated links between ND4L variants and type 2 diabetes mellitus (T2DM), where molecular dynamics simulations revealed that mutations can disrupt proton translocation pathways critical for energy production . This multifaceted approach to disease modeling allows researchers to gain insights that would be challenging to obtain directly from human studies.

What are the key differences between recombinant ND4L expression and native protein in mitochondrial studies?

Understanding the distinctions between recombinant and native ND4L is critical for experimental design and data interpretation. Several important differences must be considered:

• Post-translational Modifications:

  • Native ND4L undergoes specific modifications within the mitochondrial environment

  • Recombinant systems may lack the enzymes necessary for these modifications

  • This can affect protein folding, stability, and functional properties

• Membrane Environment Effects:

  • Native ND4L exists in the specialized lipid composition of the inner mitochondrial membrane

  • Recombinant expression often places the protein in artificial membrane environments

  • The table below compares different membrane mimetics used in recombinant studies:

• Subunit Interactions:

  • In native systems, ND4L functions within the complete complex I assembly

  • Recombinant expression often isolates the protein from its natural partners

  • Co-expression with interacting subunits (particularly ND6) improves functional relevance

• Experimental Strategies for Bridging the Gap:

  • Membrane scaffold proteins can improve membrane integration of recombinant ND4L

  • Techniques like biogenic photosystem approaches can help localize the protein to appropriate membrane domains

  • Using native-like lipid compositions during reconstitution enhances functional properties

When designing experiments with recombinant ND4L, researchers should carefully consider which aspects of native function they need to preserve and select expression and reconstitution systems accordingly.

How do mutations in ND4L affect proton translocation and energy production across species?

Mutations in ND4L have profound effects on proton translocation and energy production, with both common mechanisms and species-specific responses observed across different organisms:

• Universal Mechanisms of Dysfunction:

  • Disruption of proton channels through the transmembrane domain

  • Alterations in critical hydrogen bond networks essential for proton movement

  • Reduced coupling efficiency between electron transport and proton pumping

  • Molecular dynamics simulations reveal that mutations like M47T and C69W interrupt water molecule passage through the transmembrane region

• Species-Specific Adaptations and Vulnerabilities:

  • Ustilago maydis possesses alternative respiratory components that can partially compensate for complex I dysfunction:

    • External NADH dehydrogenase (NDH-2) that contributes significantly to NADH oxidation

    • Alternative oxidase (AOX) that can bypass cytochrome components when inhibited

  • Mammalian systems typically lack these alternative pathways, making them more vulnerable to ND4L mutations

• Tissue-Specific Effects in Higher Organisms:

  • In humans, ND4L mutations primarily affect tissues with high energy demands

  • LHON (associated with mutations like T10663C/Val65Ala) primarily affects retinal ganglion cells

  • This tissue specificity is likely due to varying energy requirements and antioxidant capacities

• Bioenergetic Consequences:

  • Reduced ATP production proportional to the severity of proton translocation disruption

  • Increased reactive oxygen species generation due to electron leakage

  • Compensatory upregulation of glycolysis and other ATP-generating pathways

  • Potential membrane potential collapse in severe mutations

• Mutation Context Effects:

  • Haplogroup background influences the expression of mutations

  • In human studies, concurrent mutations (10609T>C and 10663T>C) showed cumulative pathogenic effects

  • The same principle may apply in fungal systems, where genetic background could modulate mutation impact

Understanding these cross-species effects provides a foundation for developing interventions that might restore energy production in the presence of ND4L mutations. The study of U. maydis can be particularly valuable for identifying compensatory mechanisms that might be therapeutically induced in human patients with mitochondrial disorders.

What are common challenges in purifying recombinant ND4L and how can they be addressed?

Purification of recombinant ND4L presents several technical challenges due to its hydrophobic nature and involvement in multi-subunit complexes. The following strategies address common obstacles:

• Protein Aggregation and Inclusion Body Formation:

  • Challenge: High hydrophobicity leads to aggregation during expression

  • Solutions:

    • Lower induction temperature (16-20°C) to slow folding

    • Fusion partners (MBP, SUMO, or thioredoxin) to enhance solubility

    • Co-expression with chaperones (GroEL/GroES system)

    • Directed evolution of expression host to tolerate membrane protein overproduction

• Detergent Selection and Optimization:

  • Challenge: Harsh detergents may solubilize effectively but denature the protein

  • Solutions:

    • Screen detergent panel (DDM, LMNG, LDAO) for optimal extraction

    • Use detergent mixtures (e.g., DDM/CHS) for improved stability

    • Consider native nanodiscs for detergent-free purification

    • Employ GFP-fusion monitoring to rapidly assess folding in different detergents

• Low Yield and Purity:

  • Challenge: Membrane proteins typically express at lower levels

  • Solutions:

    • Multi-step purification combining affinity tags with ion exchange

    • Size exclusion chromatography to separate oligomeric states

    • Consider specialized expression systems like those used for biogenic photosystems

    • Optimize cell disruption methods for efficient membrane fraction recovery

• Functional Verification:

  • Challenge: Ensuring purified protein retains native activity

  • Solutions:

    • Reconstitution into liposomes with defined lipid composition

    • Activity assays measuring electron transfer or proton translocation

    • Structural verification through circular dichroism or limited proteolysis

    • Thermal shift assays to assess proper folding

For Ustilago maydis ND4L specifically, lessons can be drawn from successful approaches used with other membrane proteins. For example, the strategy of using backbone proteins like NuoK to anchor recombinant proteins to the inner membrane has shown promise for expression of membrane-bound electron transport components . Careful attention to lipid environment during purification and storage is critical for maintaining functional properties of the isolated protein.

How can researchers distinguish between ND4L activity and other NADH dehydrogenase activities in Ustilago maydis?

Distinguishing ND4L-associated activity from other NADH dehydrogenase activities in Ustilago maydis requires selective inhibitors and careful experimental design. Effective approaches include:

• Pharmacological Inhibitor Profiling:

  • Selective Inhibitors:

    • Rotenone specifically inhibits complex I but not the alternative NADH dehydrogenase (NDH-2)

    • Flavone compounds selectively inhibit NDH-2 but not complex I

    • The combination allows separation of the two activities

  • Inhibitor Concentrations:

    • Titration curves with each inhibitor can quantify the contribution of each pathway

    • In U. maydis, NDH-2 contributes approximately equally to complex I in NADH oxidation

• Substrate Specificity Analysis:

  • NADH vs. NADPH:

    • Complex I primarily utilizes NADH

    • Alternative dehydrogenases may have different cofactor preferences

  • Membrane Permeability:

    • External NDH-2 can oxidize exogenous NADH

    • Complex I only accesses matrix NADH in intact mitochondria

• Genetic Approaches:

  • Targeted Gene Disruption:

    • Knockout or knockdown of ND4L to measure remaining NADH oxidation

    • Similar approach for NDH-2 to isolate complex I activity

  • Complementation Studies:

    • Expression of recombinant ND4L in ND4L-deficient strains

    • Rescue of respiratory defects confirms functional contribution

• Analytical Methods:

  • Spectrophotometric Assays:

    • Monitor NADH oxidation at 340 nm under different inhibitor conditions

    • Track downstream electron acceptor reduction (e.g., ubiquinone at 275 nm)

  • Oxygen Consumption Measurements:

    • Clark-type electrode or Seahorse XF analyzer with selective substrates

    • Specific inhibitor addition during measurement reveals component contributions

• Experimental Workflow Example:

In U. maydis, studies have shown that the external NDH-2 contributes as much as complex I to NADH-dependent respiratory activity, making this distinction particularly important . Unlike plant NDH-2, the U. maydis enzyme is not regulated by Ca²⁺, providing another distinguishing characteristic when performing comparative studies .

What computational tools are most effective for modeling ND4L structure and dynamics?

Computational modeling of ND4L structure and dynamics requires specialized tools optimized for membrane proteins. The most effective approaches include:

• Homology Modeling Software:

  • MODELLER: Particularly effective when using templates with >30% sequence identity

  • I-TASSER: Performs well with difficult membrane proteins using threading approaches

  • AlphaFold2: Recent advances in AI-based structure prediction show promise for membrane proteins

  • For ND4L specifically, using complex I structures from related organisms as templates improves model quality

• Molecular Dynamics Simulation Packages:

  • GROMACS: Optimized for membrane systems with specialized force fields

  • NAMD: Excellent scalability for long simulations needed to capture conformational changes

  • AMBER: Offers specialized lipid parameters for mitochondrial membrane mimetics

  • Simulation times of at least 100 ns are recommended to observe relevant conformational changes and water movement

• Membrane Protein-Specific Tools:

  • CHARMM-GUI: Facilitates building membrane-protein systems with appropriate lipid compositions

  • MemProtMD: Automated pipeline for inserting proteins into simulated membranes

  • PPM server: Predicts optimal protein positioning in membranes

• Analysis Software for MD Trajectories:

  • VMD: Visualization and analysis of water channels and hydrogen bond networks

  • MDAnalysis/MDTraj: Python libraries for quantitative analysis of simulation results

  • ProDy: Specialized for analyzing protein dynamics and conformational changes

• Simulation Parameters for Optimal Results:

Practical implementation strategies include:

• Using enhanced sampling methods (metadynamics or replica exchange) to explore conformational space efficiently

• Focusing on specific regions like the predicted proton translocation pathway

• Performing comparative simulations of wild-type and mutant proteins to identify functional changes

• Calculating energy profiles for proton movement along predicted pathways

These computational approaches have been successfully applied to study mutations in ND4L, revealing how specific changes like M47T and C69W can interrupt proton translocation pathways through alterations in hydrogen bonding patterns and water channel accessibility .