Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
J Biol Chem. 2011 Jul 1;286(26):23476-88. doi: 10.1074/jbc.M110.202960. Epub 2011 May 3.
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.
纯化的哺乳动物支链α-酮酸脱氢酶复合物（BCKDC），能催化支链α-酮酸的氧化脱羧作用，其组成成分二氢硫辛酰胺脱氢酶（E3）几乎完全缺失。E3 的缺失与人类 BCKDC 亚基结合域（hSBDb）与 hE3 结合的低亲和力有关。在这项工作中，hSBDb 与哺乳动物丙酮酸脱氢酶复合物的 E3 结合域（E3BD）的序列比对表明，hSBDb 在位置 118 处有一个精氨酸，而 E3BD 处有一个天冬酰胺。用天冬酰胺取代 Arg-118 ，使得 R118N hSBDb 变体（命名为 hSBDb*）与 hE3 的结合亲和力提高了近 2 个数量级。与野生型 hSBDb 相比，结合反应的焓变为放热反应。这种更高的亲和力相互作用使得 hE3/hSBDb复合物的晶体结构能够解析至 2.4-Å 分辨率。结构表明，Arg-118 的存在造成了独特的、可能是空间和/或静电的不兼容性，这可能会阻碍 E3 与野生型 hSBDb 的相互作用。与 E3/E3BD 结构相比，hE3/hSBDb结构的界面面积更小。溶液 NMR 数据分别证实了 hE3 与野生型 hSBDb 的 Arg-118 和 Asn-118 以及突变体 hSBDb的相互作用。NMR 结果还表明，hSBDb 与 hE3 之间的界面从 hSBDb 到 hSBDb没有明显变化。综上所述，我们的结果为解释长期以来的谜团提供了一个起点，即 BCKDC 的 E2b 核心与其他α-酮酸脱氢酶复合物相比，与 E3 的结合强度要弱得多。
