The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

Abstract

The branched-chain ␣-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 ؋ 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.The mammalian mitochondrial branched-chain ␣-keto acid dehydrogenase (BCKD) 1 complex catalyzes the rate-limiting step in the oxidation of branched-chain amino acids leucine, isoleucine, and valine (1). Genetic defects in this macromolecular multienzyme complex result in the accumulation of branched-chain amino acids leading to inheritable maple syrup urine disease. The clinical phenotype includes early onset and often fatal acidosis, neurological derangement, and mental retardation among survivors (1). The mammalian BCKD complex is organized around a cubic core of 24-meric dihydrolipoyl transacylase (E2b), to which multiple copies of branched-chain ␣-keto acid decarboxylase (E1b), dihydrolipoamide dehydrogenase (E3), BCKD kinase (abbreviated as BCK), and BCKD phosphatase are attached through ionic interactions (2-4). The activity of the BCKD complex is tightly regulated by a phosphorylation/dephosphorylation cycle in response to dietary and hormonal signals (5). Phosphorylation of Ser-292 and Ser-302 in the ␣ subunit of E1b by BCK results in the inactivation of the BCKD complex (6).The structures of rat BCK in the apo-, ADP-bound, and ATP␥S-bound forms were recently determined (7). The BCK structures feature a nucleotide-binding (K) domain and a fourhelix bundle (B) domain, which are similar to modules found in bacterial protein-histidine kinases (8) and the related pyruvate dehydrogenase kinase 2 (PDK2) of the mitochondrial pyruvate dehydrogenase complex (PDC) (9). The K domain is highly conserved between BCK and members of the GHL ATPase family (10), with the presence of characteristic N1, G1, F, and G2 boxes and an "ATP lid." In BCK, the direct back-to-back interaction of two opposing K domains produces a d...

Tissue-specific and Nutrient Regulation of the Branched-chain α-Keto Acid Dehydrogenase Phosphatase, Protein Phosphatase 2Cm (PP2Cm)

“…6B). These data indicate that PP2Cm and BCKDK have potentially overlapping binding motifs within the LBD plus C-terminal hinge domain of the E2 (29). This may explain their mutually exclusive interaction with the BCKD complex.…”

“…The PP2Cm transcription is repressed by starvation or low BCAA condition, opposite from the changes observed for the BCKDK (19,20). Whereas PP2Cm binds to BCKD E2 and E1 subunits in a BCKA-independent manner, its binding motifs overlap with the BCKDK binding domain on E2 (29,30). This competition in BCKD binding is dynamically regulated by the BCKA levels.…”

“…Within the BCKD holoenzyme, E1 and E3 subunits bind to E2 through the subunit binding domain (30). BCKDK interacts with E2 via the LBD-linker domain (29). More structural analysis would be needed to define the binding motifs between PP2Cm and the E2 and the molecular basis for the sensitivity to BCKA.…”

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

“…Wild-type and mutant apoE1b proteins were obtained by exhaustive dialysis in the presence of 0.2 mM EDTA. C-terminally His 6 -tagged LBD (residues 1-84 of the E2b subunit) was expressed (16) and lipoylated in vitro with DL-6,8-thioctic acid using LplA, a lipoic-protein ligase, as described earlier (17).…”

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

“…SUMO-BDP was eluted using a 0 -600 mM NaCl gradient in the same buffer. The C-terminal His 6 -tagged wild-type and mutant E2b domain constructs were produced as described previously (23). Site-directed mutagenesis was carried out using the QuikChange kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions.…”