Investigation of Protein‐Protein Interactions in the Pyruvate Dehydrogenase Complex from Escherichia coli by Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

The pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli is a molecular machine consisting of multiple copies of three enzyme components: pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p), and dihydrolipoyl dehydrogenase (E3p), which cooperate with each other to perform the oxidative decarboxylation of pyruvate to acetyl‐CoA. To probe the protein‐protein interactions and conformational changes between these components, we used hydrogen/deuterium exchange mass spectrometry (DXMS). Based on our study, the regions of interactions are mapped as follows: 1. The interaction of E2p with the active regions of E1p that were missing from previous X‐ray crystallography (an inner loop formed by residues 401–413, outer loop formed by residues 541–557 and N‐terminal residues 1–55); 2. The binding loci on peripheral‐subunit binding domain (PSBD) of E2p that interact with E1p and E3p; 3. The interaction of the E3p redox‐active region with E1p and E2p. This DXMS method has proved to be fast, accurate and reliable in characterizing the interaction of components in PDHc. The authors thank NIHGM‐050380 for financial support.