Cosgrove Michael S, Bever Katherine, Avalos Jose L, Muhammad Shabazz, Zhang Xiangbin, Wolberger Cynthia
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry. 2006 Jun 20;45(24):7511-21. doi: 10.1021/bi0526332.
Sirtuins comprise a family of enzymes that catalyze the deacetylation of acetyllysine side chains in a reaction that consumes NAD+. Although several crystal structures of sirtuins bound to non-native acetyl peptides have been determined, relatively little about how sirtuins discriminate among different substrates is understood. We have carried out a systematic structural and thermodynamic analysis of several peptides bound to a single sirtuin, the Sir2 homologue from Thermatoga maritima (Sir2Tm). We report structures of five different forms of Sir2Tm: two forms bound to the p53 C-terminal tail in the acetylated and unacetylated states, two forms bound to putative acetyl peptide substrates derived from the structured domains of histones H3 and H4, and one form bound to polypropylene glycol (PPG), which resembles the apoenzyme. The structures reveal previously unobserved complementary side chain interactions between Sir2Tm and the first residue N-terminal to the acetyllysine (position -1) and the second residue C-terminal to the acetyllysine (position +2). Isothermal titration calorimetry was used to compare binding constants between wild-type and mutant forms of Sir2Tm and between additional acetyl peptide substrates with substitutions at positions -1 and +2. The results are consistent with a model in which peptide positions -1 and +2 play a significant role in sirtuin substrate binding. This model provides a framework for identifying sirtuin substrates.
沉默调节蛋白家族由一类酶组成，这些酶在消耗烟酰胺腺嘌呤二核苷酸（NAD+）的反应中催化乙酰赖氨酸侧链的去乙酰化。尽管已经确定了几种与非天然乙酰化肽结合的沉默调节蛋白的晶体结构，但对于沉默调节蛋白如何区分不同底物的了解相对较少。我们对与单一沉默调节蛋白——嗜热栖热菌（Thermatoga maritima）的Sir2同源物（Sir2Tm）结合的几种肽进行了系统的结构和热力学分析。我们报告了Sir2Tm的五种不同形式的结构：两种形式分别与处于乙酰化和未乙酰化状态的p53 C末端尾巴结合，两种形式分别与源自组蛋白H3和H4结构化结构域的假定乙酰化肽底物结合，还有一种形式与类似于无酶蛋白的聚丙二醇（PPG）结合。这些结构揭示了Sir2Tm与乙酰赖氨酸N末端的第一个残基（位置-1）和乙酰赖氨酸C末端的第二个残基（位置+2）之间以前未观察到的互补侧链相互作用。等温滴定量热法用于比较Sir2Tm野生型和突变型之间以及在位置-1和+2处有取代的其他乙酰化肽底物之间的结合常数。结果与一个模型一致，即肽的位置-1和+2在沉默调节蛋白底物结合中起重要作用。该模型为鉴定沉默调节蛋白底物提供了一个框架。
