Preliminary Study On The Interaction Mechanism Between Pyruvate Dehydrogenase And Dihydrolipoamide Acetyltransferase Of E. Coli

Pyruvate dehydrogenase complex(PDC) is a hub of cell energy metabolism which catalyzes pyruvate made in glycolysis to acetyl-CoA. It comes into tricarboxylic acid cycle(TCA) as the initial substrate, and offering energy by oxidative phosphorylation. In the overall complex, pyruvate dehydrogenase is the key rate-limiting enzyme. So it is most important to find out the mechanism of pyruvate dehydrogenase(E1) interaction with dihydrolipoamide acetyltransferase(E2).This experiment, at first, achieves N-terminal domain(NTD), peripheral subunit-binding domain(PSBD) and related proteins by cloning ntd and psbd etc.to prokaryotic expression systemand, expression and purification. Then, means of pull down and isothermal titration calorimetry(ITC) are used to study protein-protein interaction in vitro. Finally, in order to make mechanism clear, we determinate structure of protein NTD and its complex with PSBD by means of nuclear magnetic resonance(NMR). Now, the results achieved in the experiment are as follows:(1) we had achieved three His-tag proteins of NTD, E1 and E56, and three removal tag proteins of PSBD, HPSBD and LIPO where were from after constructing prokaryotic expression vectors, expression and purification. All the obtained proteins were identified by mass spectra and it proved that they all were correct target proteins.(2) Using pull down and ITC to research protein-protein interaction in vitro, it suggested that PSBD was only binding to NTD. LIPO did not interact with either NTD or PSBD, which meant that it was impossible to participate in the interaction of E1 and E2. HPSBD, unlike PSBD, could’t combine with NTD. At last, it compared with the thermodynamic difference of PSBD binding to NTD and E1 via ITC data.(3) Conformation of PSBD in different solution studied by means of circular dichroism(CD) and dynamic light scattering(DLS), indicating that structure of PSBD changed easily, and usually transformed the folded conformation into random coil combined without E1 and E3, which provided the structure basis for PSBD binding to E1 or E3 in turn.(4) We had obtained the solution structure of NTD with NMR, which consisted two ?-helix and random coil in two-side terminal. According to checking result of PROCHECK software, the structure had a high reliability. The complex of NTD with PSBD wasn’t accomplished.All the data supported structure and theory basis for revealing the mechanism of E1 interaction with E2, and was helpful to drug of antibiotic design based on interaction surface.