Recombinant Ornithorhynchus anatinus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Definition and Basic Properties
NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) from Ornithorhynchus anatinus is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase, commonly referred to as Complex I. This protein plays a crucial role in the minimal assembly required for catalytic activity within the complex. The primary function of MT-ND3 involves facilitating the transfer of electrons from NADH to the respiratory chain, with ubiquinone serving as the immediate electron acceptor for the enzyme system . The recombinant form of this protein is produced through molecular cloning and expression techniques, making it available for detailed biochemical studies and functional characterization.
Evolutionary Significance
The platypus (Ornithorhynchus anatinus) represents a unique evolutionary position as a monotreme mammal, exhibiting characteristics of both mammalian and reptilian ancestry. This distinctive evolutionary status makes its mitochondrial proteins, including MT-ND3, particularly valuable for comparative studies of mitochondrial function across vertebrate lineages. Analysis of MT-ND3 across primates has revealed considerable evolutionary variation, with specific positions showing high variability (Ind3 values) along the evolutionary timeline . This evolutionary plasticity provides important context for understanding the functional constraints and adaptations of mitochondrial proteins across species.
Protein Structure and Domains
MT-ND3 in Ornithorhynchus anatinus is characterized by specific structural elements critical to its function within Complex I. A particularly important structural feature is the loop located between transmembrane helices 1 and 2 (residues 24-54), which contains functionally significant amino acids . This region includes a cysteine residue at position 39 that is exposed during active mitochondrial respiration and is considered essential for the reversible transition between catalytically active and inactive states of Complex I . The dynamics of this loop influence the exposure of Cys39 and consequently affect the active-inactive state transition of the complex.
Interaction with Complex I Components
MT-ND3 functions within a highly integrated network of proteins comprising Complex I. The protein's functional significance is underscored by its interactions with multiple other subunits, forming a cohesive molecular machine for electron transfer. These interactions are critical for the assembly, stability, and catalytic function of Complex I as a whole, with disruptions potentially leading to impaired energy production and associated pathologies.
Protein-Protein Interaction Profile
Analysis of the MT-ND3 protein interaction network reveals extensive connectivity with other components of the mitochondrial respiratory chain. According to STRING database analysis, MT-ND3 demonstrates strong functional associations with multiple protein partners, achieving an average node degree of 9.82 and a high local clustering coefficient of 0.982 . This indicates that MT-ND3 participates in a tightly connected functional module within the mitochondrial proteome, emphasizing its integration within the respiratory machinery.
Key Functional Partners
The predicted functional partners of MT-ND3 include several critical components of Complex I and the wider respiratory chain:
| Protein | Description | Interaction Score |
|---|---|---|
| NDUFV2 | NADH:ubiquinone oxidoreductase core subunit V2 | 0.999 |
| NDUFS3 | NADH:ubiquinone oxidoreductase core subunit S3 | 0.999 |
| NDUFV1 | NADH_4Fe-4S domain-containing protein | 0.999 |
| NDUFS7 | NADH:ubiquinone oxidoreductase core subunit S7 | 0.999 |
| MT-ND5 | NADH-ubiquinone oxidoreductase chain 5 | 0.999 |
| MT-ND4 | NADH-ubiquinone oxidoreductase chain 4 | 0.999 |
| MT-ND2 | NADH-ubiquinone oxidoreductase chain 2 | 0.999 |
These high confidence interactions (all scoring 0.999) demonstrate the integral role of MT-ND3 within the Complex I assembly and the coordinated function of the mitochondrial respiratory chain .
Comparative Analysis Across Species
Mitochondrial genes, including MT-ND3, exhibit interesting patterns of conservation and variation across evolutionary lineages. Analysis of MT-ND3 across primates has revealed that certain positions within the protein show remarkable variability. Specifically, positions 34 and 45 have been identified as highly variable along primate evolution (with evolutionary rate indices Ind3 of 1.442 and 0.882, respectively), having accumulated 20 and 15 non-synonymous substitutions coding for eight and six different amino acids, respectively . This evolutionary plasticity contrasts with the pathogenicity of specific mutations at these same positions in humans, highlighting the complex relationship between sequence variation and functional impact.
Disease-Associated Variations
In humans, specific variations in MT-ND3, such as m.10158T>C (S34P) and m.10191T>C (S45P), are confirmed disease-associated mutations linked to Leigh Disease . While serine is the only residue encoded at these positions in normal human MT-ND3, the platypus and other species show considerable variation at these same sites. Notably, among the multiple non-synonymous substitutions affecting codon 34 of MT-ND3 across species, 10 resulted in radical changes in amino acid physicochemical properties, affecting up to seven different properties . These findings suggest that the functional impact of specific amino acid substitutions is context-dependent and may vary across evolutionary lineages.
Recombinant Protein Production and Purification
The production of recombinant MT-ND3 from Ornithorhynchus anatinus typically involves expression in mammalian cell systems, similar to other mitochondrial proteins from this species . The purification process often achieves protein purities exceeding 85%, as determined by SDS-PAGE analysis, making the recombinant protein suitable for various biochemical and structural studies . Proper storage and handling of the recombinant protein are critical for maintaining its integrity and functional properties, with recommendations including storage at -20°C to -80°C with the addition of glycerol (typically 50%) to prevent freeze-thaw damage .
Functional Rescue Strategies
Recent research has demonstrated promising approaches for addressing mitochondrial defects associated with MT-ND3 mutations. One innovative strategy involves re-engineering techniques to deliver mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes . This approach has shown success in partially restoring protein levels, complex I function, and ATP production in cells with MT-ND3 variants, suggesting potential therapeutic applications for mitochondrial disorders . While these studies have focused on human MT-ND3 variants, the methodologies could potentially be adapted for comparative studies using the platypus protein.
MT-ND3 Mutations and Mitochondrial Disorders
Mutations in MT-ND3 are associated with several mitochondrial disorders in humans, particularly Leigh syndrome and mitochondrial complex I deficiency . Specific variants, such as m.10197G>C and m.10191T>C, have been shown to significantly lower MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduced ATP synthesis . These pathogenic effects highlight the critical role of MT-ND3 in maintaining proper mitochondrial function and energy production.
Comparative Pathology and Evolutionary Medicine
The study of MT-ND3 variants across species, including Ornithorhynchus anatinus, provides valuable insights into the relationship between sequence variation and functional impact. The observation that positions which are invariant in humans (and where mutations cause disease) show considerable variation in other species suggests complex evolutionary dynamics in mitochondrial gene evolution . This comparative approach enhances our understanding of the functional constraints on mitochondrial proteins and the context-dependent effects of specific amino acid substitutions.
Advances in Mitochondrial Gene Therapy
Recent research has demonstrated promising results in addressing mitochondrial defects through innovative gene therapy approaches. The use of codon-optimized nuclear expression of mitochondrial proteins, including MT-ND3, with appropriate mitochondrial targeting sequences, has shown potential for functional rescue of mutant phenotypes . These advances represent a significant step toward potential therapeutic interventions for mitochondrial disorders, with implications extending beyond human medicine to comparative and evolutionary biology.
Evolutionary Insights and Comparative Mitochondrial Biology
The study of MT-ND3 across species, including the platypus, continues to provide valuable insights into mitochondrial evolution and function. The observation that disease-associated invariant positions in humans show considerable variation in other species highlights the complex relationship between sequence conservation, functional constraints, and evolutionary adaptation . Future research directions may include detailed comparative analyses of MT-ND3 structure and function across diverse species, potentially revealing novel insights into mitochondrial biology and the evolution of energy metabolism systems.
Product Specs
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Target Background
Function: A core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It catalyzes electron transfer from NADH through the respiratory chain, utilizing ubiquinone as an electron acceptor. This subunit is essential for the catalytic activity of Complex I.
KEGG: oaa:808710
STRING: 9258.ENSOANP00000024990
Q&A
What is Recombinant Ornithorhynchus anatinus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)?
Recombinant Ornithorhynchus anatinus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) is a laboratory-produced version of the mitochondrial protein encoded by the MT-ND3 gene from the platypus (Ornithorhynchus anatinus). This protein is a critical subunit of Complex I in the mitochondrial electron transport chain. The recombinant form allows researchers to study this mitochondrial component outside its native environment and investigate its structural and functional properties in isolation or in reconstituted systems . The protein can be produced with tags (such as His-tags) to facilitate purification and experimental manipulation, similar to other recombinant mitochondrial proteins .
How does MT-ND3 contribute to mitochondrial function?
MT-ND3 functions as an integral component of NADH dehydrogenase (ubiquinone), also known as Complex I, which is the largest of the five complexes in the electron transport chain. This complex has an L-shaped structure consisting of a hydrophobic transmembrane domain and a hydrophilic peripheral arm containing redox centers and the NADH binding site. MT-ND3 is particularly hydrophobic and forms part of the core transmembrane region of Complex I . The protein plays a crucial role in the proton-pumping mechanism that contributes to the electrochemical gradient necessary for ATP synthesis. MT-ND3 contains specific structural elements, including transmembrane helices and functional loops, that are essential for proper Complex I assembly and operation .
What is the amino acid composition and size of MT-ND3?
The MT-ND3 protein in mammals typically consists of approximately 115 amino acids and has a molecular weight of about 13 kDa . While the exact sequence for Ornithorhynchus anatinus MT-ND3 is not provided in the search results, we can compare it to the related Oncorhynchus kisutch (Coho salmon) MT-ND3, which has 116 amino acids with the sequence: "MNLITTIITITITLSAVLATVSFWLPQISPDAEKLSPYECGFDPLGSARLPFSLRFFLIAIILFLLFDLEIALLLPLPWGDQLNTPTLTLVWSTAVLALLTLGLIYEWTQGGLEWAE" . The platypus version would likely have both conserved regions essential for function and species-specific variations reflecting evolutionary adaptation.
What experimental systems are used to produce Recombinant MT-ND3?
Recombinant MT-ND3 is typically produced in prokaryotic expression systems, with E. coli being the most common host organism. For example, the Recombinant Oncorhynchus kisutch MT-ND3 was expressed in E. coli with an N-terminal His-tag to facilitate purification . For the platypus version, similar expression systems would likely be employed. The recombinant protein is often purified using affinity chromatography (leveraging the His-tag), followed by additional purification steps if needed. The final product is commonly supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE . Researchers should be aware that repeated freezing and thawing is not recommended, and working aliquots should be stored at 4°C for up to one week, with long-term storage at -20°C/-80°C .
How can researchers use MT-ND3 to investigate mitochondrial disorders?
MT-ND3 is implicated in several mitochondrial disorders, making it a valuable target for disease research. Variants of MT-ND3 are associated with Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Leigh's syndrome (LS), and Leber's hereditary optic neuropathy (LHON) . Researchers can use recombinant MT-ND3 to:
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Perform in vitro functional assays to assess the impact of specific mutations on protein activity
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Study protein-protein interactions within Complex I
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Develop antibodies for detection of MT-ND3 in patient samples
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Establish model systems to test potential therapeutic approaches
Recent research has employed molecular dynamics (MD) simulations to investigate how specific mutations in MT-ND3, such as Ser34Pro and Thr35Pro, affect protein structure and function. These mutations were found to significantly alter the flexibility of a critical loop in MT-ND3 (residues 24-54) that contains Cys39, a residue exposed during active mitochondrial respiration and thought to be necessary for the reversible transition between catalytically active and inactive states .
What techniques are most effective for analyzing MT-ND3 structural dynamics?
Researchers investigating MT-ND3 structural dynamics employ several complementary techniques:
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Molecular Dynamics (MD) Simulations: MD simulations have revealed that mutations like Ser34Pro and Thr35Pro affect loop flexibility in the MT-ND3 protein. The root mean square fluctuation (RMSF) profiles of heavy atoms in the loop (residues 40-50) were higher for these pathogenic mutations compared to wild-type, while the first part of the loop (residues 24-40) became slightly more rigid .
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3D Dynamic Representation: This technique allows visualization of structural changes caused by mutations. For example, the loss of essential interactions between loop residues and nearby subunits (such as between residues 129 of MT-ND1 and 49 of MT-ND3, and between residues 76 of MT-ND6 and 48 of MT-ND3) caused by the Thr35Pro mutation has been visualized using this method .
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Comparative Analysis: Comparing different mutations (e.g., Ser34Pro vs. Ser34Phe) can provide insights into why some variants are pathogenic while others are benign. For instance, Ser34Phe and Ser34Tyr display flexibility profiles similar to wild-type, whereas Ser34Pro shows significant alterations .
How do MT-ND3 variants contribute to mitochondrial heteroplasmy and disease?
Mitochondrial heteroplasmy (the presence of multiple mitochondrial DNA variants within a single cell) has significant implications for disease manifestation. Research analyzing over 1800 whole genome sequencing data from four Alzheimer's disease cohorts identified that MT heteroplasmy was present throughout the entire MT genome for blood samples, but was detected only within the MT control region for brain samples .
What computational approaches help predict the pathogenicity of MT-ND3 variants?
Several computational tools have been developed to predict the pathogenicity of mitochondrial variants, including those in MT-ND3:
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APOGEE 2: This tool assigns pathogenicity scores to variants. For example, the m.10191T>C (Ser34Pro) variant in MT-ND3 was classified as a Variant of Uncertain Significance (VUS) with a score of 0.51 (probability = 0.59) by APOGEE 2, despite being confirmed as pathogenic in other studies .
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Molecular Dynamics Simulations: MD simulations can predict structural and functional consequences of mutations. For the MT-ND3 protein, simulations revealed that pathogenic mutations like Ser34Pro and Thr35Pro significantly alter the flexibility of a critical functional loop, while benign variants (Ser34Phe and Ser34Tyr) exhibited wild-type-like flexibility profiles .
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Database Integration: Tools integrating data from resources like MITOMAP and ClinGen help contextualize novel variants based on known pathogenic mutations. For instance, the m.10158T>C (p.Ser34Pro) variant is reported as "confirmed" by MITOMAP and as "pathogenic" in ClinGen, associated with Leigh disease or MELAS syndrome .
What evolutionary insights can be gained from studying platypus MT-ND3?
The platypus (Ornithorhynchus anatinus) represents a unique evolutionary lineage of mammals, making its mitochondrial genes, including MT-ND3, valuable for comparative evolutionary studies. Interesting evolutionary features of MT-ND3 include:
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Untranslated Extra Nucleotide: In MT-ND3 genes from many species of birds and turtles, there is an extra nucleotide that is not translated to protein. This feature is managed either through translational frameshifting or RNA editing to maintain the functionality of the reading frame. This extra nucleotide feature in turtles suggests they might be related to Archosauria, as evidenced by molecular phylogeny studies .
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Conservation of Functional Domains: Despite evolutionary divergence, certain functional domains in MT-ND3, particularly those involved in Complex I assembly and function, show conservation across species, highlighting their essential nature.
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Species-Specific Variations: Comparing MT-ND3 sequences across species can identify regions under different selective pressures, potentially reflecting adaptations to different metabolic demands or environmental conditions.
The study of platypus MT-ND3 can provide insights into the evolution of mitochondrial function in early mammalian lineages and improve our understanding of the relationship between structure and function in this essential component of cellular energy production.
How does Ornithorhynchus anatinus MT-ND3 compare to MT-ND3 from other species?
While detailed comparative data specifically for Ornithorhynchus anatinus MT-ND3 is not provided in the search results, we can infer potential comparisons based on related information:
Across species, MT-ND3 maintains its core function as part of Complex I, but species-specific variations reflect evolutionary adaptations. These differences make comparative studies valuable for understanding both conserved functional domains and regions under distinct selective pressures. The availability of recombinant proteins from different species facilitates such comparative research approaches.
What are the key challenges in working with Recombinant MT-ND3?
Researchers working with Recombinant MT-ND3 face several technical challenges:
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Protein Stability: As a highly hydrophobic membrane protein, MT-ND3 can be difficult to maintain in a stable, properly folded state outside its native environment. Storage recommendations typically advise avoiding repeated freeze-thaw cycles and keeping working aliquots at 4°C for limited periods (up to one week) .
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Functional Reconstitution: Assessing the functional properties of MT-ND3 requires its incorporation into a lipid environment or reconstitution with other Complex I subunits, which can be technically challenging.
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Post-translational Modifications: Recombinant expression systems may not reproduce all the post-translational modifications present in the native protein, potentially affecting certain functional studies.
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Species-Specific Differences: When using platypus MT-ND3 as a model for human mitochondrial diseases, researchers must account for species-specific differences that may affect the interpretation of results.
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Mutation Analysis: Determining the pathogenicity of MT-ND3 variants can be complicated by discrepancies between computational predictions and experimental findings. For example, the m.10191T>C variant was classified as a VUS by APOGEE 2 despite being confirmed as pathogenic in other studies .
How can researchers optimize studies of MT-ND3's role in Complex I assembly and function?
To optimize studies of MT-ND3's role in Complex I:
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Combined Structural and Functional Approaches: Integrate structural studies (e.g., MD simulations) with functional assays to correlate structural changes with functional impacts. This approach has successfully revealed how mutations in MT-ND3's loop region affect its flexibility and potentially its role in Complex I activation/inactivation .
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Site-Directed Mutagenesis: Introduce specific mutations to examine the importance of key residues like Cys39, which is exposed during active mitochondrial respiration and is thought to be necessary for the reversible transition between catalytically active and inactive states of Complex I .
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Interaction Studies: Investigate MT-ND3's interactions with other Complex I subunits, particularly MT-ND1 and MT-ND6, as disruptions to these interactions by mutations (e.g., Thr35Pro) have been implicated in pathogenicity .
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Comparative Analysis of Variants: Study both pathogenic and benign variants affecting the same residue (e.g., Ser34Pro vs. Ser34Phe and Ser34Tyr) to understand the molecular basis of pathogenicity .
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Integration with Clinical Data: Correlate experimental findings with clinical presentations of patients carrying MT-ND3 mutations to establish genotype-phenotype relationships.
