title: Structural and thermodynamic basis for weak interactions between dihydrolipoamide dehydrogenase and subunit-binding domain of the branched-chain alpha-ketoacid dehydrogenase complex.
abstract: The purified mammalian branched-chain α-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain α-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other α-ketoacid dehydrogenase complexes.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. chad.brautigam@utsouthwestern.edu
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.forename: R Max
authors.initials: RM
authors.lastname: Wynn
authors.forename: Jacinta L
authors.initials: JL
authors.lastname: Chuang
authors.forename: Mandar T
authors.initials: MT
authors.lastname: Naik
authors.forename: Brittany B
authors.initials: BB
authors.lastname: Young
authors.forename: Tai-Huang
authors.initials: TH
authors.lastname: Huang
authors.forename: David T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D013329:Structure-Activity Relationship
chemical_list: D008058:Dihydrolipoamide Dehydrogenase