长春花RLK1样受体激酶（CrRLK1L）是受体激酶（Receptor like kinase, RLK）蛋白超家族的亚家族之一。CrRLK1L家族成员具有两个特征性的胞外Malectin结构域。CrRLK1L家族成员在植物繁殖、生长、免疫和环境响应等过程中均发挥重要作用。快速碱性化因子（RAPID ALKALINIZATION FACTOR, RALF）是一类新发现的植物肽类激素家族，研究表明其是CrRLK1L受体激酶成员如FER、ANX1/2的配体。拟南芥LRE及LRE-LIKE GPI-ANCHORED PROTEIN1/2/3 (LLG1/2/3) 家族是一类胞外GPI膜锚定蛋白，被报道参与了CrRLK1L-RALF信号通路。然而，RALF是如何被CrRLK1L感知的以及RALF被CrRLK1L感知是否还需要其他蛋白（如LLG）的参与，这其中的分子机制还不清楚。在本论文中，我们使用晶体学手段解析了apo-FERECD, apo-ANX1ECD, apo-ANX2ECD, apo-LLG1ECD,与 RALF23-LLG2-FERECD复合物共五个结构，并结合体外生化实验，阐明了植物肽类激素RALF被受体激酶-膜锚定蛋白这一异型受体复合物识别的分子机制。本论文的研究表明，GPI蛋白LLG1/2/3可作为受体，直接识别配体RALF23，并在RALF23的诱导下发生明显的构象变化，形成新的作用面以结合受体激酶FER，使FER-LLG1/2/3这一异型受体复合物共同完成对配体RALF23的识别。本项研究还纠正了植物Malectin也会识别多糖的认识，证明了动植物Malectin结构域在分别进化后，已获得了截然不同的配体识别功能。有别于过去研究发现的受体激酶同型识别配体的传统模式，本论文研究揭示的受体激酶与膜锚定蛋白形成异型复合物识别配体的模式，为植物受体激酶以及GPI膜锚定蛋白的结构功能研究提供了全新的范例。此外，本论文还提供了几个晶体学结构解析实例，包括使用单波长反常散射法解析蛋白质和核酸晶体结构、分子置换法中结构未知区域的从头搭建和孪晶的解析、化学小分子的构建与修正，希望能够为晶体学初学者提供借鉴意义。
Receptor kinases (RKs) of the Catharantus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment.Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides have been proposed as ligands for several CrRLK1L members. The mechanistic basis of this perception, however, is unknown. Here, we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LRE-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1, which is genetically important for RALF23 responses, or the related LLG2, directly binds RALF23 to nucleate the assembly of a RALF23-LLG1/2-FER heterocomplex. A conserved N-terminal region of RALF23 is sufficient for its biochemical recognition by LLG1/2/3, and binding assays suggest other RALFs sharing this conserved N-terminal region may be perceived in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1/2/3. Our work reveals an unexpected mechanism of plant peptide perception by GPI-anchored proteins in concert with a phylogenetically-unrelated RK, which provides a molecular framework to understand how diverse RALF peptides may regulate multiple processes through perception by distinct heterocomplexes between CrRLK1L RKs and GPI-anchored proteins of the LRE/LLG family.This thesis also provides several examples of crystal structure determination, including the solutions of protein and nucleic acid crystal using single-wavelength anomalous dispersion method, ab initio building of unknown structure and solution of twinning crystal using molecular replacement method, building and refinement of small chemical molecules, hoping to be of reference to beginners in bio-crystallography.
