Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

Journal Article · Mon Feb 27 04:00:00 EST 2012 · J. Biol. Chem.

DOI:https://doi.org/10.1074/jbc.M110.202960· OSTI ID:1029839

Brautigam, Chad A; Wynn, R Max; Chuang, Jacinta L; Naik, Mandar T; Young, Brittany B; Huang, Tai-huang; Chuang, David T ^[1]

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1. AS

The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-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-{angstrom} 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 {alpha}-ketoacid dehydrogenase complexes.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: FOREIGNOTHERNIH
• OSTI ID: 1029839
• Journal Information: J. Biol. Chem., Journal Name: J. Biol. Chem. Journal Issue: (26) ; 07, 2011 Vol. 286; ISSN JBCHA3; ISSN 0021-9258
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
AFFINITY
ARGININE
ASPARAGINE
CALORIMETRY
CRYSTAL STRUCTURE
DECARBOXYLATION
ELECTROSTATICS
ENTHALPY
MUTANTS
OXIDOREDUCTASES
RESOLUTION
SPECTRA
THERMODYNAMICS
TITRATION
WEAK INTERACTIONS