中国科学院昆明动物研究所机构知识库

小鸟苷三磷酸酶(small guanosine triphosphatases)的蛋白超家族成员，也被称为小G蛋白、小GTPases或Ras超家族，它们是一类重要的功能蛋白，主要介导细胞因子、生长因子和多种细胞外信号通路，对细胞的生长、分化、存活、增殖等多种功能的调节发挥重要作用。尽管小G蛋白家族各成员间存在差异，但是它们也都具有一些功能的特征：（1）都是单体小G蛋白，能与GTP或GDP结合，并且拥有高度保守的GTP催化结构域；（2）该家族中的大多数成员是癌基因，当被异常激活后会导致细胞无限增殖，引起肿瘤；（3）家族中大多数成员都有内源性的GTP酶活性，将GTP水解为磷酸（Pi）和GDP；（4）家族中的大多数成员C-末端都可以通过翻译后的脂酰化修饰与膜结合。基于序列和功能相似性分析，目前真核生物中的小G蛋白主要分为六个亚家族：Ras、Rho、Rab、Ran、Arf和RJL。由于小G蛋白家族成员大多数与癌症的发生相关，因此对其家族成员的研究一直都是研究的热点。我们解析了小G蛋白家族中RJL亚家族的第一个晶体结构：非洲爪蟾Rbj全长的晶体结构，非洲爪蟾Rbj与家族中的其他成员具有高度的同源性。从结构上看Rbj主要包括两个结构域，N端的催化结构域（G-domain）和C端的DnaJ结构域。在晶体的堆积方式上，Rbj是以二聚体的方式存在，整体结构类似“三角形”，并且Rbj蛋白C端的DnaJ结构域比较灵活，对Rbj的功能有重要影响。在得到晶体结构的基础上，我们开始探讨影响Rbj活性的结构机理。其N端催化结构域具有小G蛋白家族成员催化结构域的典型特征，包括五个α螺旋和六个β折叠组成的催化核心区。但是与家族中其他大多数成员不同的是，虽然Rbj具有家族中保守的催化核心结构域，但是其天然不具有水解GTP的能力。通过对结构的分析我们发现有至少三种可能的原因：催化反应核心位点缺乏保守的谷氨酰胺提高了抑制水解反应发生时的过渡态能量；水解核心位置的组氨酸可能会阻止水分子向GTP的靠近，而这步是达到过渡态所必需的；同时switch II区与GTP结合的构象非常稳定，可以防止水解过程中所需的构象变化。在体外酶活实验中，验证了对活性影响的六个关键氨基酸，分别是Ala25、Glu26、Tyr48、His75、Pro76和Phe77。圆二色谱实验也证明了Rbj处于GTP结合状态下蛋白最稳定。同时我们解析的Rbj蛋白结构是小G蛋白家族中第一个含有DnaJ结构域的晶体结构，而之前的研究证实DnaJ结构域可以调控Hsp70的活性，因此通过进一步研究，我们提出了一种Rbj与Hsp70可能的作用方式。另一部分研究，我们尝试组装并结晶解旋酶家族成员Prp2与其辅助因子Spp2蛋白复合物。大多数真核基因被内含子打断，必须将内含子从pre-mRNA中去除掉才能进行正常的基因表达，这一过程主要由一个称为剪接体的复合物完成。剪接体的组成和结构都是高度动态的，8个DExD/H家族RNA解旋酶在控制剪接体的构象重排方面发挥着重要作用。其中在剪接体的构象重排过程中DEAH-box蛋白Prp2（一个RNA依赖的ATP酶），其在第一步剪接反应前的剪接体Bact复合物向有催化活性的B*复合物的转变过程中发挥了重要的作用。同时Prp2的辅助蛋白Spp2可以增强其ATP酶活性，帮助其发挥正常的功能。因此研究Prp2与Spp2蛋白的相互作用对理解解旋酶家族成员在剪接体中的功能非常重要，也为其他解旋酶与辅助因子的相互作用提供一个可参考的结果。通过实验，成功的在不同缓冲液和盐浓度条件下组装了Prp2与Spp2全长复合物以及Prp2与Spp2截短体的蛋白复合物，并在不同温度条件下都进行了晶体筛选，为后续的进一步研究打下了坚实的基础。本研究有助于人们进一步认识小G蛋白家族，尤其是新发现的RJL亚家族。结构解析进一步阐明了RJL亚家族成员Rbj天然不具有水解GTP能力的原因。而且目前的研究发现Rbj蛋白与一些癌症的发生相关，因此对Rbj的结构解析也为相关抗癌药物的设计从结构上提供了重要的信息。同时对解旋酶家族成员Prp2与其辅助蛋白Spp2复合物的组装，也为理解旋酶家族成员在剪接体中的功能提供了可参考的信息。

Members of the protein superfamily of small guanosine triphosphatases, also known as small G-proteins, small GTPases or the Ras superfamily, which is a kind of important functional proteins, mainly mediated cytokines, growth factors and a variety of extracellular signaling pathways, and play an important role in the regulation of cell growth, differentiation, survival and proliferation. Despite the differences among the members of the small G protein family, they also have some functional characteristics: (1) Both are small monomeric G proteins that bind to GTP or GDP and possess a highly conserved GTP catalytic domain; (2) Most members of the family are oncogenes, which, when abnormally activated, causing tumors; (3) Most members of the family have endogenous GTPase activity that hydrolyzes GTP into phosphoric acid (Pi) and GDP; (4) The C-terminus of most members of the family can bind to the membrane through post-translational lipid acylation modification. Based on sequence and functional similarity analysis, eukaryotes were divided into six subfamilies: Ras, Rho, Rab, Ran, Arf and RJL. The majority of small G protein superfamily members are related to the occurrence of cancer, so the research on the small G protein superfamily members has always been a hot spot in the scientific community.We have determined the first crystal structure of the RJL subfamily in the small G protein family: the full-length crystal structure of Xenopus laevis Rbj, it is highly homologous to other members of the family. Structurally, Rbj mainly includes two domains, a N-terminal catalytic domain and a C-terminal DnaJ domain. In the way of crystal packing, Rbj exists as a dimer, and the overall structure is similar to "triangle", and the DnaJ domain at the C-terminus of Rbj protein is flexible and has an important influence on the function of Rbj. Based on the crystal structure, we began to explore the structural mechanism of Rbj activity. Its N-terminal catalytic domain has the typical characteristics of the small G protein superfamily member catalytic domain, including the catalytic core region composed of five alpha helices and six beta sheets. but unlike most other members of the family, it naturally does not have the ability to hydrolyze GTP. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine at the core of the catalytic region raises the energy of the transition state inhibiting hydrolysis. The histidine may restrain the waters from moving closer to the GTP, a step that is required for attain the transition state. They also stabilize the GTP-bound conformation of switch Ⅱ and could prevent conformational changes required during hydrolysis. In the in vitro enzyme activity experiments, six key amino acids affecting the activity were verified, namely Ala25, Glu26, Tyr48, His75, Pro76 and Phe77, respectively. Circular dichroism experiments also demonstrated that Rbj is most stable in the GTP-bound state. At the same time, the structure of Rbj is the first crystal structure containing the DnaJ domain in the small G protein family. Previous studies have confirmed that the DnaJ domain can regulate the activity of Hsp70. Therefore, through further study, we proposed a possible interaction mode between Rbj and Hsp70.In another part, we try to assemble and crystallize the helicase family member Prp2 and its cofactor Spp2 protein complex. Most eukaryotic genes are interrupted by itrons, which must be excised from pre-mRNA for proper gene expression. This process is primarily performed by a complex called the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. During conformational rearrangement of the spliceosome, DEAH-box protein Prp2( an RNA-dependent ATPase) facilitates the remodeling of the spliceosomal Bact complex to the catalytically activated B* complex just before step one of splicing. At the same time, the accessory protein Spp2 of Prp2 can enhance its ATPase activity and help it perform its normal function. Therefore, studying the interaction between Prp2 and Spp2 proteins is important for understanding the function of the helicase family members in the spliceosome, and also provides a reference for the interaction of other helicase-cofactors. Through experiments, the full-length Prp2 and Spp2 complexes and the protein complexes of Prp2 and Spp2 truncation were successfully assembled under different buffer and salt concentrations, and the crystals were screened under different temperature conditions, laying a solid foundation for further reaearch. This study is helpful for further understanding of the small G protein superfamily, especially the newly discovered RJL subfamily. And further structural analysis shows the reason why the RJL subfamily member Rbj does not have the ability to hydrolyze GTP naturally. Moreover, current research has found that Rbj protein is associated with the occurrence of some cancers, so the structural analysis of Rbj also provides important information for the design of related anticancer drugs. At the same time, the assembly of the helicase family member Prp2 and its accessory protein Spp2 complex provides information for better study of the helicase family members.