Backbone Dynamics of the Lipoyl Acid-Bearing Domain of Human mitochondrial Branched-Chain Alpha-Ketoacid Dehydrogenase - A NMR 15N{H}-Relaxation Study .



Hui-Ting Chou1, Chi-Fon Chang2 and Tai-Huang Huang1,2

1Department of Physics, National Taiwan University, Taipei, Taiwan2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan


The E2 component of human mitochondrial Branched-chain alpha-ketoacid dehydrogenase (BCKD) complex is a homo-24-meric dihydrolipoyl transacylase of molecular mass over 1.1 x 106 daltons. The Lipoyl acid-Bearing Domain (LBD) of the E2 component has 87 amino acid residues. Mutations in LBD are related to the maple syrup urine disease (MSUD). Our NMR structure showed that it comprises two four-stranded beta-sheets forming a flattened beta-barrel. We further apply 15N NMR relaxation techniques to probe the backbone dynamics of LBD. Total of 73 each of the R1, R2, and 15N{H} NOE were determined at 500 MHz and 600 MHz. These data were analyzed by model-free formalism and reduced spectral density mapping, assuming that chemical shift anisotropy (CSA) and dipolar interactions are the only two primary relaxation mechanisms. The overall rotational correlation time (τm), the generalized order parameters (S2), the correlation times of local motion (τe), and the spectral density functions ( J(0.87ωH), Japp(0), and J(ωN) ) were extracted, assuming axially symmetric diffusion tensor for LBD. When the motions are mapped to the structure we found that the loop regions exhibit slow conformational exchange motion in microsecond/ millisecond timescale while the backbone β-strand regions are rather rigid. As expected, the terminal regions show larger amplitude of fast motion in picosecond/nanosecond time scale. The lipoylation site, Lysine 44, is exposed to the solvent and located in a turn connecting two beta-strands. This site is found to be flexible.