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首页> 外文期刊>Journal of Molecular Biology >Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.
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Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

机译：NMR T（2）弛豫实验揭示了蛋白质间的相互作用：脂肪嗜热芽孢杆菌的丙酮酸脱氢酶多酶复合物的脂酰结构域和E1组分。

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T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled. Copyright 2000 Academic Press.
机译：T（2）弛豫实验与化学位移和定点诱变数据相结合，用于鉴定与嗜热脂肪芽孢杆菌丙酮酸脱氢酶多酶复合物中弱但特异的蛋白质-蛋白质相互作用有关的位点。丙酮酸脱羧酶成分，异四聚体E1（alpha（2）beta（2）），负责第一个承诺的和不可逆的催化步骤。与二氢脂酰乙酰基转移酶（E2）组件相连的脂酰的伴随的还原性乙酰化涉及E1和E2多肽链的脂酰结构域之间的弱，短暂但特异的相互作用。游离脂酰结构域（9 kDa）和游离E1alpha（41 kDa），E1beta（35 kDa）和完整的E1alpha（2）beta（2）（152 kDa）组件之间的相互作用，所有基因或亚基因的产物使用异核2D NMR光谱研究了在大肠杆菌中表达的C-γ-转移酶。用均一的（15）N标记的脂酰结构域和未标记的E1组分进行实验。在存在和不存在E1（alpha（2）beta（2））或其单独的E1alpha或E1beta组件的情况下，从骨架（15）N自旋自旋弛豫时间的变化确定了脂酰域上的主要接触点。尽管E1alpha亚基包含与必需辅因子硫胺素二磷酸相关的序列基序，但脂酰结构域的识别分布在E1alpha和E1beta的位点上。脂酰结构域上的单点突变（N40A）大大降低了其被同源E1还原乙酰化的能力。但是，N40A突变域似乎与E1相互作用，类似于野生型域。这表明，由于化学位移数据表明，也许是由于脂酰结构域结构中的轻微扰动，尤其是在脂酰赖氨酸β-转角区域中，N40A脂酰结构域的脂酰基未以正确的方向呈现给E1。 。与E1的相互作用和随后的还原性乙酰化不一定是耦合的。版权所有2000学术出版社。

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