【作者】 刘合军；

【导师】 牛立文； 滕脉坤；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 Nit蛋白质家族广泛分布于动物、植物、真菌和原核细胞中,是氰水解酶超家族的第十分支。哺乳动物具有两种Nit蛋白,分别为Nitl和Nit2,它们之间具有很高的序列同源性。酵母Nit2是哺乳动物肿瘤抑制因子Nitl的同源蛋白,与Nitl一样,其底物特异性均是未知。近期研究发现,哺乳动物推测的肿瘤抑制因子Nit2是可以特异性水解α-酮戊二酸单酰胺和α-酮基琥珀酰胺的ω-酰胺酶。目前,对哺乳动物Nit2的催化机制了解甚少,而且哺乳动物Nitl的自然底物也是未知的。为此,我们解析了酵母Nit2蛋白分别在其活性中心与α-酮戊二酸和草酰乙酸共价结合的复合体结构。结构分析表明,酵母Nit2通过其Arg173、 Thr196和Thr199这三个氨基酸残基识别并结合α-酮戊二酸和草酰乙酸的α-端官能团,并通过其高度保守的催化三联体CEK结合α-酮戊二酸和草酰乙酸的γ/β-端。另外,酵母Nit2高度保守的Phe195与配体α-端官能团存在一种新型的γ/β型阴离子-π分子识别作用。同时,通过酵母Nit2结构分析发现,其二聚化参与催化反应进程。酵母Nit2的β6/7-发夹可能通过二聚体界面的传递作用而参与这一催化反应进程。酵母Nit2中参与配体相互作用的残基在Nit家族蛋白中具有高度的序列保守性,这暗示了该机制可能为Nit蛋白通用的识别与催化机制。不过,我们发现酵母Nit2与哺乳动物Nit2在结合口袋处也有重要差异,这导致了其以α-酮戊二酸单酰胺作为底物时的ω-酰胺酶活性极低,类似于哺乳动物的Nitl的活性。通过分析酵母Nit2活性口袋的大小,推测其可能结合较大的底物分子。本研究中所解析的酵母Nit2C169S突变体与未知内源性配体结合的复合物结构表明,酵母Nit2的自然底物可能是α-酮戊二酸单酰胺-N-酰胺取代二肽或者类似大小的其他α-酮戊二酸单酰胺-N-酰胺取代衍生物。哺乳动物Nitl在口袋大小方面与酵母Nit2类似,它可能具有非常相似的底物结合特异性。本研究对于酵母Nit2酶活机制的研究加深了我们对哺乳动物Nit2蛋白质酶催化机制的了解,同时为探索哺乳动物Nit1的自然底物进而解释其生物学功能提供了线索。酵母Hif1最早是在核内的组蛋白B型乙酰化酶复合体中发现的。虽然它不参与乙酰化酶活性的调控,但参与B型乙酰化酶调节的端粒沉默并能促进核小体装配。现己发现,Hifl作为组蛋白的伴侣分子发挥其生物学功能。它与组蛋白伴侣Asfl在染色质重装配方面具有同等重要的作用,在复制偶联和非复制偶联的的染色质重装配过程中,可能同样起到招募组蛋白结合复合物结合到染色质上发挥相应功能的作用。此外,其人源同源蛋白核自身抗原精子蛋白(NASP)参与诸如细胞增殖等重要的生命调控活动。为此,我们解析了组蛋白伴侣分子Hifl的晶体结构。结构分析表明,Hifl是由一个TPR结构域和一个插入的酸性无规卷曲肽段组成。TPR结构域为Hifl提供基本的结构骨架,插入的肽段结合在TPR结构域上并可能改变TPR结构域的构象。我们发现,Hifl通过结合组蛋白H3/H4而结合核心组蛋白八聚体。在这个结合过程中,Hifl中的插入肽段起到重要的作用。另外,基于三维结构的蛋白质空间特征比对暗示,Hifl中TPR结构域的凹槽可能起到结合肽段的作用。分子对接计算表明,TPR结构域的凹槽可能起到结合组蛋白H3的尾巴的作用,而插入肽段可能起到识别组蛋白H4尾巴的修饰状态的作用。本研究解析的Hifl晶体结构目前是SHNi-TPR家族中第一个被解析的结构,这为了解SHNi-TPR家族蛋白质的基本特征提供重要的结构基础。同时,酵母Hifl晶体结构的解析,为今后深入研究核内组蛋白B型乙酰化酶及其与组蛋白的相互作用提供初步的结构分析模型。更多还原

【Abstract】 Nit protein family, as the branch10of nitrilase superfamily, is widely distributed in animals, plants, fungi and prokaryotic cells. Mammals have two Nit proteins, namely Nitl and Nit2, which have highly conserved amino acid sequences. Yeast Nit2is a homologue of mammalian tumor-suppressor protein Nitl whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2, a putative tumor-suppressor, is identical to be an ω-amidase, an enzyme that catalyszes the specific hydrolysis of a-ketoglutaramate and a-ketosuccinamate. However, the enzymatic mechanism of mammalian Nit2is ambiguous while the natural substrates of mammalian Nitl haven’t been found out. Here, we solved crystal structures of yeast Nit2in complex with a-ketoglutarate and oxaloacetate, which were thought to be the reaction intermediates of the hydrolysis, respectively. These structures reveal that Arg173, Thr196and Thr199of yeast Nit2could bind the a-moiety of the intermediate and the highly conserved canonical catalytic triad CEK could bind the γ/β-group. In addition, highly conserved Phe195of yeast Nit2interacts with a-moiety via a newly founded η6type anion enhanced π-π interaction. Structural analysis indicates that the dimerization of yeast Nit2is important to the putative catalysis process. β6/7-Hairpin lid may trigger the catalytic process via the dimer interface. The catalytic process should be a general mechanism of Nit proteins because of the highly conserved residues involved in the catalysis and dimerization. However, the active site of yeast Nit2has important differences comparing to mammalian Nit2, resulting in extremely low specific activity towards a-ketoglutaramate, which is similar to mammalian Nitl. The volume of the binding pocket of yeast Nit2is big enough for large molecular binding. We have solved the crystal structure of yeast inactive Nit2C169S mutant protein in complex with an unknown endogenous ligand. The structure reveals that the natural substrate of yeast Nit2may be a kind of dipeptide-N-substituted analogues of a-ketoglutaramate or other similar molecules. Mammalian Nitl may have similar pocket property comparing to yeast Nit2, which means they may have similar substrate specificity. Our study of yeast Nit2would not only expend our knowledge of the catalytic mechanism of mammalian Nit2, but provide a clue for exploring the natural substrate of mammalian Nitl then understanding its biological role in the cell. Yeast Hifl was first found to be a nonessential component of nuclear B-type histone acetyltransferase complex (HAT-B). It participates in telomeric silence and promotes the precipitate of nucleosome, although it has no effect on the specific activity of HAT-B complex. In present view, as a histone chaperone protein, Hifl is essential as protein Asfl that recruits histone binding protein to the chromatin in the process of replication-coupled or-independent chromatin reassembly. In addition, nuclear autoantigenic sperm protein (NASP), human homologue of yeast Hifl, involves in many vital important life regulation progress such as cell proliferation. For the importance of Hifl, we solved its crystal structure. The structural analysis of Hifl reveals that it consists of a TPR domain and an interrupted acid loop. The TPR domain consisting of4TPRs constructs the basic structural framework of Hif1, while the interrupted acid loop binds the TPR domain and may change the conformation of the TPR skeleton. In this study, we found that Hifl could bind to histone octamer via binding to H3/H4tetramer. The acid loop is important for this kind of binding. In addition, structure-based alignment of Hifl indicates that the cave shaped by TPR domain may bind with specific peptides. According to the molecular docking experiment, the cave of TPR domain could be inosculated by histone H3tail, while the acid loop may recognize the post-translational modification state of H4tail. The crystal structure of Hifl, the first structure of SHNi-TPR family, provides insight into the structural characterization of SHNi-TPR proteins. Our research of yeast Hifl provides a preliminary structural model for our further study of the interaction between HAT-B complex and histone complex.更多还原