Potential Impact of Phosphorylation on Malate Dehydrogenase and Citrate Synthase Protein‐Protein Interaction

Abstract

Fine tuning of metabolic protein pathways is an insightful way to learn about the progression of diseases linked to metabolism, such as Alzheimer’s disease. Analyzing specific protein interactions in a structure‐function relationship, opens the discussion for potential therapeutic interventions. Malate Dehydrogenase (MDH) and Citrate Synthase (CS) interact in human mitochondria forming a metabolon found within the Krebs Cycle. While the interaction between MDH and CS is not novel, the complete interface between MDH and CS is not yet determined. Furthermore, how these interactions may be modulated by post‐translational modification has yet to be investigated. This study attempts to investigate if key residues driving isoform specific interactions between the MDH and CS are regulated by phosphorylation. Published docking models of MDH and CS highlight several potential sites of interaction, but the key residues of MDH in the interface are not experimentally determined. We performed additional docking predictions using Hawkdoc and SwissDock with published cross‐linked lysine residues of MDH‐CS. Interaction between cytosolic MDH and CS is reported to be much weaker, thus a primary structure alignment highlights the areas of unique residues that might be critical for mitochondrial MDH‐CS interactions. Meta‐analysis of phosphorylation databases from mammalian cells/tissues have identified several sites of phosphorylation with unknown function. To examine the key sites of interaction between MDH and CS and its regulation, we selected several putative phosphorylation sites specific to mitochondrial MDH; S45, T85, S222, and T224, and generated phosphomimic S/T to D mutants. Utilizing his‐tagged recombinant human MDH and CS, we measured interaction using fast thermal melt assays with the presence and absence of crowding agents (10% glycerol; 20% PEG) to determine regulation of phosphomutant interactions. Lastly, we conducted pull‐down assays to illustrate the possible changes in interactions with CS in the wild‐type and phosphomimic MDH‐CS pairs. In determining the interface and post‐translational modification between MDH and CS with these key residues, we can clarify where and how the governing mechanism regulates MDH and CS interaction. This will enable us to gain insight into the modulation of the binding interface interaction and contribution towards altering various metabolic disease pathways.