Protein-Protien Interaction
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In cells, proteins perform various functions and interact with each other in different ways. These interactions play a crucial role in regulating enzyme activity, inducing structural changes, and controlling cellular processes. One type of protein interaction is the formation of supramolecular complexes called metabolons. These complexes involve sequential metabolic enzymes that combine to share reactants and products, promoting efficient metabolic pathways.
Metabolons were first described as "substrate channeling" by Dr. Paul Srere in 1985. These complexes were observed in carbohydrate metabolism, where proteins interacted due to limited water availability. The aggregation of proteins within metabolons decreased the need for solvation by water, thus reducing the surface area required for water-protein interactions. Substrate channeling within metabolons allowed for the direct transfer of intermediates between sequential enzymes, increasing reaction rates and isolating metabolites from competing side reactions.
Organized systems of sequential proteins in a metabolic pathway create local enrichments of shared products, facilitating efficient reactions without relying on the random diffusion of metabolites. This organization also protects unstable intermediates, limits the half-life of reactive molecules, and sequesters potentially harmful metabolites. Examples of well-known protein complexes include the pyruvate dehydrogenase complex and the tricarboxylic acid (TCA) cycle metabolons in mammalian systems. Similar metabolons have been identified in other organisms, including plants.
Research on metabolons and protein-protein interactions extends to various organisms and metabolic pathways. These interactions are critical for normal cellular function, and dysregulation can contribute to disease. Non-metabolic protein complexes, known as protein "interactomes," control important physiological processes and represent potential targets for drug development, especially in modulating protein-protein interactions.
Mitochondrial MDH interacts with several proteins within the mitochondria, forming functional complexes that contribute to various metabolic processes. One prominent interaction is with citrate synthase (CS), another enzyme in the tricarboxylic acid (TCA) cycle. MDH and CS form a supramolecular complex that facilitates the efficient transfer of substrates and products between the two enzymes, enhancing the metabolic flow within the TCA cycle. This interaction is crucial for maintaining the balance of metabolites and sustaining energy production in the mitochondria.The mitochondria house the mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex, which is an integral component of the Krebs tricarboxylic acid (TCA) cycle metabolon. This metabolon facilitates sequential reactions within the cycle without the need for diffusion of intermediates into the bulk matrix. The complex is known to play a dynamic role in regulating the TCA cycle by enhancing metabolic flux. Recent research utilizing Microscale Thermophoresis analysis focused on the porcine heart MDH-CS complex, revealing that the association of the complex is influenced by the substrates and products of the MDH and CS reactions. Specifically, NAD+ and acetyl-CoA, the substrates, were found to enhance complex association, while NADH and citrate, the products, weakened the affinity of the complex. Furthermore, it was observed that oxaloacetate enhanced the interaction only in the presence of acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS, favoring the MDH-CS association. Other metabolic factors, such as ATP and low pH, were also found to enhance the complex association. These findings indicate that the dynamic formation of the MDH-CS multi-enzyme complex is modulated by various metabolic factors responding to respiratory metabolism, potentially contributing to the feedback regulation of the TCA cycle and adjacent metabolic pathways.
In addition to the MDH-CS complex, supramolecular assembly of enzymes into metabolon structures is believed to facilitate efficient transport of reactants between active sites via substrate channeling. Notably, recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were studied in vitro and found to adopt a homogeneous native-like metabolon structure. To investigate the role of specific residues, site-directed mutagenesis targeted highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites. Among the mutations, the CS(R65A) variant retained high catalytic efficiency. However, it was observed that substrate channeling between the CS mutant and mMDH was severely impaired, resulting in a significant decrease in the overall channeling probability from 0.99 to 0.023. These findings provide direct mechanistic evidence for the channeling of reaction intermediates within the metabolon structure. Disruption of this interaction could have significant implications for the control of flux in central carbon metabolism.
Cytosolic MDH is predicted to interact with several proteins within the cytosol, forming functional complexes that contribute to various metabolic processes. One such interaction is with glutamate oxaloacetate transaminase (GOT), also known as aspartate aminotransferase (AAT). MDH and GOT are enzymes involved in amino acid metabolism, and their interaction plays a role in the interconversion of malate and oxaloacetate, as well as the transfer of amino groups between different amino acids. This interaction could help to coordinate amino acid metabolism and maintain metabolic homeostasis in the cytosol. Cytosolic MDH has been found to interact with malic enzyme 1 (ME1), an enzyme involved in malate metabolism. The interaction between MDH and ME1 modulates the flux of malate through different metabolic pathways, allowing for the integration of various metabolic processes and maintaining metabolic flexibility in the cytosol. Another protein believed to interact with cytosolic MDH is phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in gluconeogenesis. The interaction between MDH and PEPCK could contribute to the regulation of glucose production by facilitating the conversion of oxaloacetate to phosphoenolpyruvate. This interaction is important for maintaining glucose homeostasis and energy balance in the cytosol. The understanding of their interaction is primarily based on the known metabolic pathways and the interconnectedness of these enzymes. PEPCK is responsible for the conversion of oxaloacetate to phosphoenolpyruvate, a crucial step in gluconeogenesis. On the other hand, MDH catalyzes the interconversion of malate and oxaloacetate in the TCA cycle. Since the metabolite oxaloacetate is a common intermediate between PEPCK and MDH, it is reasonable to suggest their potential interaction to facilitate the efficient transfer of oxaloacetate between these enzymes.
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Determine specific sites of interaction between MDH and CS
Impact of allosteric regulators on interaction
Impact of phosphorylation on interactions
Human cytosolic MDH binding partners
Parasitic MDH interactions
Information & Resources
BioGrid (Database of Protein, Genetic and Chemical Interactions: hMDH2)
STRING (Protein-Protein Interaction Networks: hMDH2)
IntAct (Protein Interaction Database and Analysis System: hMDH2)
MINT (The Molecular INTeraction Database: hMDH2)
Introduction to Protein Protein Interactions (handout)
Tools to examine protein-protein interaction
IP/Pulldown
Crosslinking Proteins
Relevant Publications
Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites (Bartholomae FEBS 2013)
List of Skills/ Techniques Needed: list with link to protocol pages
A list of adopters: Amy Parente, Joseph Provost, Celeste Peterson… (email josephprovost@sandiego.edu to add your name!)
