SIRT1与乙酰化底物、NAD^+或烟酰胺的相互作用

Interactions of SIRT1 with Acetyl Substrate,NAD^+ or Nicotinamide

沉默信息调节因子2相关酶1(SIRT1)是一类腺嘌呤二核苷酸(NAD+)依赖性去乙酰化酶。烟酰胺是SIRT1催化的去乙酰化反应的产物,也是SIRT1的一种非竞争性抑制剂。利用蛋白同源模建和分子对接方法构建了"SIRT1/NAD+/p53乙酰化多肽"和"SIRT1/ADPR(二磷酸腺苷核糖)/烟酰胺"的两种复合物结构模型。根据"SIRT1/NAD+/p53乙酰化多肽"的复合物模型,发现SIRT1的265位丝氨酸(S265)和346位天冬酰胺(N346)与NAD+有相互作用,275位丝氨酸(S275)位于NAD+结合口袋的入口处。通过酶动力学实验证明:将S265、N346和S275突变之后会降低甚至消除NAD+与SIRT1的相互作用。另外,根据"SIRT1/ADPR/烟酰胺"的复合物模型,烟酰胺的酰胺基团与347位异亮氨酸(I347)、348位天冬氨酸(D348)形成了氢键,同时其嘧啶环埋在由262位丙氨酸(A262)、270位异亮氨酸(I270)、273位苯丙氨酸(F273)、316位异亮氨酸(I316)和347位异亮氨酸(I347)所包围的疏水环境中。将I316突变成丙氨酸,提高了酶与烟酰胺的结合能力,也显著提高了烟酰胺的抑制活性,这一现象表明烟酰胺结合在SIRT1的C口袋处。

SIRT1 is a NAD＋-dependent protein deacetylase. Nicotinamide is a reaction product of the SIRTl-catalyzed deacetylation and also a noncompetitive inhibitor of SIRT1. Here we built the complex structures of ＂SIRT1/NAD＋/p53 acetyl peptide＂ and ＂SIRT1/ADPR/nicotinamide＂ by protein homology modeling and molecular docking. According to the docking results, $265 and N346 of SIRT1 are involved in the interactions with NAD＋ , while $275 is at the entrance of the NAD＋ binding pocket. Consistently, our enzyme kinetics studies proved that mutations of $265, $275 or N346 either reduced or destroyed the interaction of NAD＋ with SIRT1. In addition, the carboxamide group of nicotinamide forms hydrogen bonds with I347 and D348, while the pyridine ring is buried in a hydrophobic environment surrounded by A262, I270, F273, I316 and I347. Interestingly, mutation of I316 to alanine increased the affinity of nicotinamide and dramatically improved its inhibitory activity, indicating that nicotinamide binds in the C pocket of SIRT1.