【作者】 徐进新；

【导师】 刘劲松；

【作者基本信息】 中国科学技术大学 ， 结构生物学， 2012， 博士

【摘要】 Sorting nexins(SNXs)是一类含有SNX-PX结构域,并在膜泡运输中发挥重要作用的蛋白。PX结构域能与磷脂酰肌醇结合,并介导含有PX结构域的蛋白与富含特定磷脂酰肌醇的膜结合。目前在哺乳动物中已经发现了33种SNXs,有些SNXs只含有PX结构域；有些除了含有PX结构域还含有其他结构域。根据结构域的组成,SNXs可以分为：SNXPX、SNXPX-BAR和SNXPX-other。Sorting nexin16(SNX16)属于SNXPX-other,它除了含有PX结构域外,在C端还有一个Coiled coil结构域。有研究表明,SNX16既能结合初级内体也能结合次级内体,并在表面生长因子受体(EGFR)由初级内体向次级内体的运输中发挥着重要的作用。在果蝇的神经细胞中,SNX16参与突触生长受体(synaptic growth receptor)的运输和突触生长信号传递的调节。研究还表明,SNX16与水泡口炎病毒感染、膀胱癌和丙肝病毒的复制有关,SNX16可能是治疗这些疾病的潜在靶标。目前SNX16发挥功能的分子机制还不清楚,为了更好的了解SNX16执行功能的结构基础,我们开展了SNX16的结构生物学研究。我们解析了SNX16的一个片段(SNX16100-278,氨基酸从100刘278)的3.3A(空间群P21)和3.35A(空间群P212121)的晶体结构。SNX16100-278形成稳定的、剪刀状的二聚体。SNX16100-278包含PX结构域和Coiled coil结构域。二聚体中,两条链的Coiled coil结构域形成平行的左手超螺旋结构。通过结构分析和分子对接,我们发现SNX16100-278二聚体中PX结构域和Coiled coil结构域共同组成独特的PI3P结合口袋。通过将SNX16-PX和高同源性的nCISK-PX结构域进行比对分析,我们发现PX结构域中PPII/a2的构象可调节传统的磷脂酰肌醇结合口袋的大小,并进而决定PX结构域与磷脂酰肌醇结合的特异性。SNXPX是一类仅含有PX结构域的SNXs蛋白。目前认为SNX3、SNX10、 SNX11、SNX12、SNX22和SNX24都属于SNXPX。结构研究也表明,Grd19p(酵母SNX3)、SNX12和SNX22除了具有传统的PX结构域外,不含有其他的结构域。最近一些SNXPX蛋白的生物学功能也开始被研究。SNX3参与组成retromer复合物,调节Wntless的循环运输。SNX10具有诱导细胞成泡的功能。但是这些蛋白发挥功能的结构基础还不知道。进一步的研究发现,SNX10中单独的传统PX结构域并不具备诱导细胞成泡的功能,传统PX结构域下游的31个氨基酸也是SNX10发挥功能所必须的。为了探明SNX10传统PX结构域下游的31个氨基酸是否具有结构,以及SNX10传统PX结构域和下游的31个氨基酸在结构上是如何组织的。我们对SNX10和SNX10的同源蛋白-SNX11(SNX10和SNX11的氨基酸序列有52%的一致性)进行了结构生物学研究。我们没有获得SNX10的晶体,但我们获得了SNX11两个片段(SNX11-142C和SNX11-170C)的晶体结构。通过分析SNX11-170C的结构,我们发现在SNX11传统PX结构域的下游有2个α螺旋(α4和α5),这两个螺旋与上游传统PX结构域形成紧密的球状结构。据此,我们推测SNX11-170C可能代表了一个新的PX结构域,我们称之为PX-extended(PXe)结构域。通过序列分析,我们推测SNX10可能也具有这个新的PXe结构域。更多还原

【Abstract】 Sorting nexins (SNXs), a large family proteins containing SNX-PX domain, are involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols (PIs) and recruites the PX domain containing proteins to the membrane rich in those PIs. Up to date,33SNXs have been found in mammalia. According to the domain composition, those SNXs can be divided into three classes:SNXPX, SNXPX-BAR and SNXPX-other.SNX16belongs to SNXpX-other, and is proposed to contain an N terminal PX domain and a C terminal Coiled coil domain. Previous studies showed that SNX16can be recruited to early endosome and late endosome, and is proposed to regulate EGFR trafficking from early endosome to late endosome.In Drosophila melanogaster, SNX16functions to regulate synaptic growth receptor and synaptic growth signaling. Researches also showed that SNX16maybe involve in some diseases, such as vesicular stomatitis virus infection, bladder cancer and hepatitis C virus replication.Here we report the crystal structure of SNX16100-278(residues100-278) at resolution of3.3A and3.35A, in P21and P212121space group, respectively. In the crystal structure, SNX16100-278, containing PX domain and Coiled coil domain, forms a dimeric structure resembling, like a pair of shears. SNX16100-278dimer contains unique PI3P binding pocket, formed by the PX domain and the dimeric coiled coil. By comparing with the crystal structure of mCISK-PX, we found that the conformation of the PPII/a2Loop mediates the size of the conventional PI binding pocket, and it may determine the PX domain binding specificity to different PIs.SNXPX is a group of proteins that contains PX domain only. It consists of SNX3-. SNX10, SNX11, SNX12, SNX22and SNX24. The structures of Grdl9p (yeast homologue of SNX3), SNX12and SNX22have been determinated. All of these structures show the conventional PX domain only. Recently some SNXPX proteins have been investigated in various biological processes. SNX3has been reported to regulate the recycling of Wntless through a novel retromer-dependent pathway. SNX10is involved in the homeostasis of endosome and is required for primary cilia formation. However, the structural basis of these activities remains largely unknown.SNX11is a SNXPX member and highly homologous with SNX10. We started to investigate the crystal structure of SNX10and SNX11, but we only obtained SNX11 crystal. We determinated two SNX11structures, SNX11-142C (residues7-142) and SNX11-170C (residues7-170), with resolution of1.78A and1.6A, respectively. The SNX11-170C structure shows that there are two a helices (a4and α5) downstream of the conventional PX structure. The two helices interact with the preceeding conventional PX structure through hydrophobic and hydrophilic interactions. a4, a5and this conventional PX structure form a stable globular domain, and we named this domain PX-extended (PXe) domain. Amino acid sequence alignment showed that SNX10may also contain this novel PXe domain.更多还原