Howard M J, Chauhan H J, Domingo G J, Fuller C, Perham R N
Cambridge Centre for Molecular Recognition, Department of Biochemistry,University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
J Mol Biol. 2000 Jan 28;295(4):1023-37. doi: 10.1006/jmbi.1999.3391.
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.
结合化学位移和定点诱变数据进行的T(2)弛豫实验，用于确定嗜热脂肪芽孢杆菌丙酮酸脱氢酶多酶复合物中参与弱但特异性蛋白质 - 蛋白质相互作用的位点。丙酮酸脱羧酶组分是一种异源四聚体E1(α(2)β(2))，负责第一个关键且不可逆的催化步骤。与二氢硫辛酰胺乙酰转移酶（E2）组分相连的硫辛酰基伴随的还原乙酰化涉及E1与E2多肽链硫辛酰结构域之间弱的、短暂但特异性的相互作用。使用异核二维核磁共振光谱研究了游离硫辛酰结构域（9 kDa）与游离E1α（41 kDa）、E1β（35 kDa）以及完整的E1α(2)β(2)（152 kDa）组分之间的相互作用，所有这些都是在大肠杆菌中过表达的基因或亚基因的产物。实验使用均匀(15)N标记的硫辛酰结构域和未标记的E1组分进行。根据在存在和不存在E1(α(2)β(2))或其单个E1α或E1β组分时主链(15)N自旋 - 自旋弛豫时间的变化，确定了硫辛酰结构域上的主要接触点。尽管E1α亚基包含与必需辅因子硫胺二磷酸相关的序列基序，但对硫辛酰结构域的识别分布在E1α和E1β的位点上。硫辛酰结构域上的单点突变（N40A）显著降低了其被同源E1还原乙酰化的能力。尽管如此，N40A突变结构域似乎与野生型结构域类似地与E1相互作用。这表明N40A硫辛酰结构域的硫辛酰基没有以正确的方向呈现给E1，这可能是由于硫辛酰结构域结构的轻微扰动，特别是如化学位移数据所示的硫辛酰 - 赖氨酸β - 转角区域。与E1的相互作用以及随后的还原乙酰化不一定是耦合的。
