survivin

帮助癌细胞的蛋白质的结构

当癌细胞快速增生时，它们好像需要一种名为survivin的蛋白质的帮助。这种蛋白质在癌细胞中含量很丰富，但在正常细胞中却几乎不存在。癌细胞与survivin蛋白的这种依赖性使得survivin自然成为制造新抗癌药物的靶标，但是在怎样对付survivin蛋白这个问题上却仍有一些未解之谜。最近据一些研究人员报道，survivin蛋白出人意料地以成双配对的形式结合在一起——这一发现很有可能为抗癌药物的设计提供了新的锲机。

Survivin蛋白属于一类防止细胞自我破坏(即凋亡)的蛋白质。这类蛋白质主要通过抑制凋亡酶(caspases)的作用来阻碍其把细胞送上自杀的道路。以前一直没有科学家观察到survivin蛋白与凋亡酶之间的相互作用。也有其它迹象表明survivin蛋白扮演着另一个不同的角色——在细胞分裂后帮助把细胞拉开。

为了搞清survivin蛋白到底起什么作用，美国加利福尼亚州的结构生物学家Joseph Noel和同事们率先认真观察了它的三维结构。他们将X射线照射在该蛋白质的晶体上，并测量了X射线的偏转角度，这可以让研究人员计算出蛋白质中每个原子所处的位置。他们得到的结果指出，survivin蛋白形成一种结和，这是其它凋亡抑制物不形成的。这几位研究人员在7月份出版的《自然结构生物学》杂志中报告，survivin分子的一部分出人意料地与另一个survivin 分子的相应部分连结在一起，形成了一个被称为二聚物(dimer)的蛋白质对。研究人员推测这些survivin蛋白的二聚物可能在细胞分裂时维持关键的分子结构。如果这种蛋白质必须成双配对后才能发挥作用，那么用一种小分子把它们分开也许能对付癌症。

生物化学家Guy Salvesen说，掌握了survivin蛋白的结构“并没有澄清它是怎样防止细胞自杀的疑点”。但是他说，这些蛋白质配对的事实确实让人惊奇，“你几乎很难找到不重要的二聚作用区域”。他也同意两个蛋白质的接触面将是抗癌症药物集中对付的良好靶标。

Crystal structure of human Survivin bound to histone H3 phosphorylated on threonine-3.

Survivin, a subunit of the Chromosome Passenger Complex, binds the N-terminal tail of histone H3, which is phosphorylated on T3 by Haspin kinase, and localizes the complex to the inner centromeres. We used X-ray crystallography to determine the residues of Survivin that are important in binding phosphomodified histone H3. Mutation of amino acids that interact with the histone N-terminus lowered in vitro tail binding affinity and reduced CPC recruitment to the inner centromere in cells, validating our solved structures. Phylogenetic analysis shows that non-mammalian vertebrates have two Survivin paralogs, which we name Class A and B.

A distinguishing feature of these paralogs is H to R change in an amino acid that interacts with the histone T3 phosphate. The binding to histone tails of the human Class A paralog, which has a histidine at this position, is sensitive to changes around physiological pH, while Xenopus Survivin Class

B is less so. Our data demonstrate that Survivin paralogs have different characteristics of phosphospecific binding to threonine-3 of histone H3, providing new insight into the biology of the inner centromere.

crystal structure of human Survivin in complex with H3(1-10) peptide Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

CRYSTAL STRUCTURE OF SURVIVIN BOUND TO THE N-TERMINAL TAIL OF HSGO1

Localization of the chromosomal passenger complex (CPC) at centromeres during early mitosis is essential for accurate chromosome segregation and is dependent on the phosphorylation of histone H3. We report the 2.7 ? resolution structure of the CPC subunit Survivin bound to the N-terminal tail of histone H3 carrying the Thr3 phosphorylation mark (Thr3ph). The BIR domain of Survivin recognizes the Ala1-Arg2-Thr3ph-Lys4 sequence, decoding the modification state and the free N terminus of histone H3 by a strategy similar to that used by PHD fingers. The structural analysis permitted the identification of putative Survivin-binding epitopes in other mitotic proteins, including human Shugoshin 1. Using biophysical and structural data, we show that a phospho-mimic N-terminal sequence such as that of hSgo1 (Ala1-Lys2-Glu3-Arg4) contains the specificity determinants to bind Survivin. Our findings suggest that the CPC engages in mutually exclusive interactions with other constituents of the mitotic machinery and a histone mark in chromatin.

Crystal structure of Hepatitis B X-Interacting Protein at high resolution Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here were port the crystal structure of the shortest isoform of HBXIP (91 aa long,?11 kDa) at 1.5 ? resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a super family of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended ?-sheet area formed by 10 anti-parallel ?-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two ?2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is dimeric form in solution. The fit between the experimental small angle X-ray scattering curve and the back calculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.

Crystal structure of a Survivin-Borealin-INCENP core complex

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions of the core components lead to functional interdependence. Association of the core "passenger" proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization.

Crystal structure of the Borealin-Survivin complex

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction

surface identified in the crystal structures.

SOLUTION STRUCTURE OF HUMAN SURVIVIN

NMR studies of the antiapoptotic protein survivin have been used to determine the homodimer interface of the protein in solution and to identify residues of the protein that interact with Smac/Diablo. In solution, survivin(1-120) forms a bow-tie-shaped dimer whose interface is composed of its N-terminal residues as well as residues connecting its BIR domain to the C-terminal alpha helix. The solution structure resolves the controversy regarding the two possible dimer interfaces for survivin observed in X-ray crystal structures. The structural basis for the interaction between survivin and Smac/Diablo was also investigated. When Smac/Diablo or N-terminal Smac/Diablo peptide analogues are added to a solution of survivin, specific residues near alpha4 and beta3 are perturbed. NMR experiments indicate that the peptides bind across the third beta-strand of survivin in a manner similar to the way Smac/Diablo peptides bind to the BIR3 domain of X-linked IAP (XIAP).

SURVIVIN DIMER H. SAPIENS

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.

X-RAY CRYSTAL STRUCTURE OF THE HUMAN ANTI-APOPTOTIC PROTEIN SURVIVIN

Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.

suivivin即生存素，是新近发现的一种凋亡抑制基因，属于哺乳类凋亡抑制基因(inhibitor of apoptosis proteins，IAPs)家族。与lAPs家族的其他蛋白不同，suivivin不但具有抑制细胞凋亡的作用，而且参与了细瞧有丝分裂的调节和胞质分裂，与细胞增殖有关。

一方面，这些起始位点可以与一些G1期特异的反式作用因子相互作用，从而抑制相关基因在G1期的表达；另一方面，某些因子可以凋节suivivin的表达，进而发挥其调控作用。

suivivin在胚胎组织中规律性的广泛表达，考虑它参与有丝分裂纺锤体核姐妹染色单体分裂的调控。作为有丝分裂的调节者、参与者，可能和其他G2／M期检查点一道在细胞分化过程中保留了遗传的忠实性，参与细胞的生长和分化。suivivin是caspase-3和caspase-7的直接抑制因子，使caspase-3不能有效地水解微管结构蛋白，维持纺锤体的完整性。确保胚胎细胞有丝分裂正常进行。suivivin正常表达是胚胎发生、发育的前提和保障，适度的suivivin更是正确完成胚胎细胞有丝分裂和细胞增值的先决条件，而且它的表达与胚胎的质量密切相关。

Survivin即生存素，是新近发现的一种凋亡抑制基因，哺乳类lAP家族的新成员。1997年Ambmsini等用效应细胞蛋白酶受体-1(effector cell protease receptor-1，EPR-1)的cDNA在人类基因组库中筛选克隆出来的，其基因序列由3个内含子和4个外显子组成，与EPR-l的序列呈反向互补。Survivin的基因全长14．7kb，位于17号染色体q25区，靠近端粒。成熟mRNA全长1629nt，编码共有142个氨基酸、分子质量为16．5×103的胞质蛋白，人与鼠survivin蛋白的同源性为84．3％。

与其他的lAP蛋白分子相比，survivin蛋白结构是非常独特的，它是同源二聚体，N端仅含有一个相对保守的串联的杆状病毒IAP重复序列2(baculoviral lAP repeat 2，BIR2)结构域；从进化上讲，它与在酵母、线虫以及果蝇中发现的lAP的BIR结构域

更为相似，因此它有可能保留了一些较为原始的功能，其中半胱氨酸46-脯氨酸47-苏氨酸48，即Cys46-Pro47-Thr48三个氨基酸插人序列将BIR分子分成两半。BIR分子中含有对凋亡有重要作用的氨基酸残基：色氨酸67(Trp67)、脯氨酸33(Pro33)和半胱氨酸84(Cys84)，survivin通过这些残基与caspase3和caspase7结合，抑制caspases的活性。在C端缺少锌指结构，所以不能直接参与caspase的结合，而代之为一个长度为6．5nm、由42个氨基酸组成的两性a螺旋结构与细胞分裂微(microtube)蛋白结合，主要调节survivin的定位分布，而且这种结合同survivin的抗凋亡活性紧密相关。序列分析还

发现survivin蛋白包含3个潜在的蛋白激酶C(protein kinase C，PKC)磷酸化位点：苏氨酸21(Thr21)、丝氨酸88(Ser88)、和苏氨酸127(Thr127)，两个酪氨酸激酶Ⅱ的磷酸化位点：Thr48和Thr97，以及一个蛋白激酶A(protein kinase A，PKA)磷酸化位点：Ser81，这些潜在的磷酸化位点在survivin的磷酸化以及对其功能调控中的作用有待进一步的研究来确定。

生存素（Survivin）是新近发现的结构独特的凋亡抑制蛋白家族成员，具有抑制凋亡和调节细胞分裂的双重功能。它参与细胞的生长和分化，在终末分化成熟的正常成人组织中无表达，但重新表达于多种转化细胞和人类绝大多数常见肿瘤组织中，这为肿瘤治疗提供了一个新靶点。它与肿瘤的关系密切，目前研究包括survivin在各种肿瘤中的表达，survivin在肿瘤细胞中的作用机制以及在肿瘤早期诊断和治疗上的应用等。Caspase-3是一种促凋亡因子，是Caspase系统引起细胞凋亡的最下游成分。Survivin通过抑制Caspase-3的活性而阻断凋亡的发生过程。本文研究旨在通过对结直肠癌患者中凋亡抑制因子survivin的表达研究，对这一潜在的肿瘤标志物有进一步认识，就Survivin在肿瘤的早期诊断和治疗预后方面的价值探讨结直肠癌组织中Survivin表达的临床意义。

Survivin是新近被克隆出的一种凋亡抑制蛋白，是凋亡抑制蛋白（inhibitor ofapoptosis protein,IAP）家族中的一员。它功能上具有抑制细胞凋亡和调节细胞分裂的双重作用，主要在细胞周期的G2/M期表达，以特异的可饱和的方式与有丝分裂纺锤体微管结合，并受微管反应动力学的调节。Survivin的分布具有明显的组织特异性，它在正常组织中不表达，而特异性表达于人和鼠的胚胎发育组织以及多数人类肿瘤细胞。在正常成人组织的表达仅见于胸腺、睾丸和分泌期子宫内膜。

Survivin通过抑制Caspase-3的活性而阻断凋亡的发生过程。Caspase-3是一种促凋亡因子，是Caspase系统引起细胞凋亡的最下游成分，它在凋亡机制中扮演重要的角色，参与了多种因素诱导的细胞凋亡并使细胞凋亡得以完成。Caspase-3以无活性的酶原形式存在于细胞浆中，在凋亡信号的刺激下经蛋白酶水解作用切去原结构域，形成四聚体而活化，参与一系列与凋亡有关的细胞变化过程。

Survivin结构相对简单，只包含2个特定结构：N端单拷贝的BIR区域和C端疏水的a一螺旋结构。除BIR区域

外．survivin与其他已知IAP无序列同源性。BIR区域是survivin蛋白配体结合区域，片层中Thr34-Pro-Glu—Arg残基被认为是周期蛋白依赖性激酶的结合位点；而被Zn2+以配位键形式结合在一起的由Cys57、Cys60、His77、Cys84四个氨基酸残基构成的四面体对survivin抗凋亡功能很重要。a一螺旋结构主要调节survivin的定位分布，是其同微管结合所必需的，去除a一螺旋后survivin就丧失了与λ微管蛋白在纺锤体中心的定位功能，无法在微管组装中心聚集Cdk4、p21和caspase-3，进而失去了调控凋亡和细胞周期的功能。

survivin参与许多关键的细胞程序，能结合大量蛋白质形成复杂的相互作用网络。不同定位的survivin功能不同。位于线粒体膜间腔的survivin能保护细胞免于凋亡，胞浆内survivin影响着间期微管稳定性，在细胞周期调解中起重要作用。survivin通常以二聚体形式存在。二聚体结构和带负电荷的伸展表面是survivin抗凋亡和保护细胞正常二倍体所必需的，决定了survivin对细胞凋亡和有丝分裂周期具有双重调控功能。但现有证据表明有些细胞程序需要survivin单体参与，survivin单体能在体内、外与Smac／DIABLO和XIAP结合，使细胞避开caspase依赖凋亡途径，这种保护作用甚至比二聚体更显著。二聚体一单体之间平衡机制形同功能开关，决定了survivin功能的多样性和复杂性。

Survivin具有抑制凋亡和调节细胞增殖的双重作用，是迄今发现最强的凋亡抑制因子，也是目前发现的惟一与细胞周期密切关联的IAP家族成员。Survivin抑制凋亡的机制可能通过直接或间接干涉caspase的功能而抑制细胞凋亡。Survivin直接作用于caspase，主要是抑制caspase-3、caspase-7的活性，阻断细胞的凋亡过程。survivin也可通过p21间接抑制caspase。Survivin的表达具有细胞周期依赖性，Fengzhi研究发现Survivin仅在Gz／M期表达，在有丝分裂的早期，Survivin与有丝分裂纺锤体的微管有特异性的可逆性饱和反应，并受微管动力的调节，用微管抑制剂干预Survivin与微管之间的相互作用，可

以导致Survivin抗凋亡活性的丧失以及caspase-3的激活，从而引起细胞的凋亡。这个结果表明，Survivin部分地通过对纺锤体微管的作用调节细胞的有丝分裂过程，进而发挥其抗凋亡功能。

Survivin不仅具有抗凋亡作用，而且在细胞分裂时具有明显的作用。研究表明，Survivin是着丝粒相关蛋白，在细胞周期的不同阶段，Survivin的定位是变化的。研究发现，Survivin还与细胞的分化有关。Survivin的二聚体结构和环绕天冬氨酸一71的疏水界面是其抗凋亡和保持倍体性所必须的，这是Survivin既抗凋亡又调节细胞分化的双重作用的结构基础。

生存素是耶鲁大学的Altieri等于1997年用效应细胞蛋白酶受体2l(effectorcellproteasereceptor21，EPR21)cDNA在人类基因组文库中筛选克隆出来的，与其他的IAP蛋白分子相比，survivin蛋白结构是非常独特的，它是同源二聚体。从进化上讲，它与在酵母、线虫以及果蝇中发现的IAP的BIR结构域更为相似，因此它有可能保留了一些较为原始的功能。

参与细胞增殖和分裂参与血管生成抑制细胞凋亡

生存素具有独特的蝶形领结样结构, 仅含一个杆状病毒IAP 重复序列(baculoviral IAP repeat,BIR),位于生存素氨基端,为生存素发挥细胞凋亡作用所必需。羧基端不含有锌指状结构,而代之以一个含有42 个氨基酸形成的α-螺旋结构。

生存素不但具有抑制细胞凋亡的作用,而且参与细胞有丝分裂的调节和细胞质分裂。有丝分裂初期,生存素与有丝分裂纺锤体微管蛋白特异性结合,抑制Caspase 活性,降低凋亡的发生,维护有丝分裂的正常进行。干扰生存素和微管蛋白的反应,可使生存素抗凋亡活性丧失,增加Caspase-3的活性,导致细胞的G2 / M 期凋亡,细胞分裂异常,伴有多倍体细胞形成。在细胞周期的G2 / M 期,生存素能对抗异常因素诱导的细胞凋亡,而肿瘤细胞生存素的过度表达,能使细胞克服这个凋亡的调控点而利于转化细胞,完成异常的有丝分裂。生存素也可与p21蛋白结合于中心粒上,使p21聚集在中心粒上,减弱p21对细胞周期的负性调节, 使细胞周期缩短。

Structure of the core CPC in a similar orientation with a zoom-in (in stereo) showing the hydrophobic core of Borealin that replaces the Survivin dimeric counterpart

Survivin exists as a homodimer in the apo state (Figure 4A, upper panel) and as a monomer in the CPC-bound state. In the apo state, dimerization of human Survivin is mediated by the first turn of the C helix, the linker region, and the N-terminal residues of the BIR domain.

The interaction between Survivin and Borealin in the CPC exhibits striking molecular mimicry of the apo-Survivin–Survivin interaction. The hydrogen-bonding network engaging the backbone of Borealin Arg66Bor, Met68Bor,and Trp70Bor parallels that of Survivin Leu96Sur, Leu98Sur,and Gly99Sur in the apo structure. Formation of the complex involves an induced-fit rearrangement of the hydrophobic residues of Survivin, which undergo small but relevant changes in the positioning of their side chains to recognize Borealin and INCENP.

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connectedto its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A° resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions the core components lead to functional interdependence. Association of the core ‘‘passenger’’ proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization.

The N-terminal 141 residues of Borealin have been shown to interact with Survivin as efficiently as the full-length protein and to be sufficient for the localization of both Borealin and Survivin to the central spindle and midbody