Structural and thermodynamic basis for weak interactions between dihydrolipoamide dehydrogenase and subunit-binding domain of the branched-chain α-ketoacid dehydrogenase complex

Chad A. Brautigam, R. Max Wynn, Jacinta L. Chuang, Mandar T. Naik, Brittany B. Young, Tai Huang Huang, David T. Chuang

研究成果: 雜誌貢獻文章

15 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)23476-23488
頁數13
期刊Journal of Biological Chemistry
286
發行號26
DOIs
出版狀態已發佈 - 2011 七月 1

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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