Interface investigation the protein interaction interface between malate dehydrogenase and citrate synthase

Abstract

Enzymes complexes (metabolons) support the metabolic reactions through substrate channeling. Several enzymes in the Krebs cycle are found in protein‐protein metabolons including mitochondrial malate dehydrogenase (MDH2) and citric synthase (CS). While the weak transitory interaction between these enzymes has been demonstrated, the key residues of human MDH2 involved in the interface with CS has not been identified. The purpose of this study is to probe possible residues of MDH responsible for binding to CS. Because cytosolic MDH (MDH1) poorly binds to CS, we initially identified unique sequences that were involved in or adjacent to reported or predicted binding sites of MDH2 and CS. Four regions were selected and residues corresponding to the primary sequence of cytosolic MDH1 were substituted in place of mitochondrial MDH2. Computational models of all the monomeric constructs were made using phyre2 homology modeling, subsequent refinement of the monomers using Galaxy Refine and construction of the dimer forms using the appropriate template in PyMol. Dimers were then refined using Galaxy Refine Complex to give replicate structures of the final models. Potential metabolon formation was explored using HawkDock with the dimer versions of MDH and Citrate Synthase either with or without restraints imposed by published metabolon models.Wild‐type human genes of MDH1, MDH2, and CS were codon optimized for bacterial expression and cloned into the C‐terminus of each gene in a pET28a expression vector. Each MDH1‐MDH2 substitution (DS1‐DS4) were constructed by Gibson cloning. Resulting structure and functional impacts were determined by melting points and kinetic parameters (specific activity, Km, Vmax and Kcat) in purified proteins. Interactions between wild‐type MDH1/2 and CS were compared to MDH2 DS mutants. To qualitatively demonstrate interactions, pull‐down assays were performed in the absence and presence of crowding agents, glycerol, PEG or Ficoll 70. Finally wild‐type MDH 1 and 2 vs MDH2 mutants were subjected to competitive pull‐down assays to identify regions responsible for isozyme specific interactions. This work will show four of the potential interacting regions between MDH and CS and lead to a better understanding of dynamics of this metabolic pair.