The C-terminal hinge region of lipoic acid-bearing domain of E2b is essential for domain interaction with branched-chain alpha-keto acid dehydrogenase kinase.
The branched-chain alpha-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 x 10(-6) m and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK.
Chuang
Jacinta L
JL
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Wynn
R Max
RM
Chuang
David T
DT
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D008322:Mammals; D008928:Mitochondria / Q000201:enzymology; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D008063:Thioctic Acid / Q000378:metabolism*
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D008063:Thioctic Acid; D011494:Protein Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase