Site-directed cross-linking with KLK proteases from prostate

许多生物过程依赖于结合其他分子的蛋白质，这些分子通常也是蛋白质。其中一些复合物非常稳定，可以在结构生物学中进行分析。然而，也存在寿命较短的复合物，研究起来要困难得多。目前不稳定络合物的交联分子组分的程序是相当不特异性的。因此，站点定向链接的新方法是可取的。所谓的点击化学反应是在蛋白质的特定位置形成共价键，其中包括公认的1,3-偶极环加成反应，也称为叠氮-炔点击反应，以及新开发的蛋白质的硫醇-烯偶联反应。这两种方法都基于非天然或非规范氨基酸（nnAA），这些氨基酸可以通过操纵遗传密码和细胞培养中的蛋白质合成而被纳入选定的蛋白质中。蛋白质-配体相互作用的合适模型系统是来自前列腺的人钾化钾素相关肽酶（klk），特别是klk2、3、4、5和11。这些丝氨酸蛋白酶在受精过程中具有重要的生理功能，在前列腺癌等病理条件下作用发生改变。了解几种KLK晶体结构是将nnAAs放置在蛋白酶的选定位置以连接它们与其他分子的良好基础。为此，将主要使用这些klk的非活性变体，这些变体在点击反应后不会切割连接的分子。点击反应的两种方法将分两步应用：1。含有活性nnAA的非活性KLK变体分别与含有叠氮化物和炔作为nnAA反应伙伴的肽，或半胱氨酸和烯烃侧链连接。这些肽来源于天然底物，前列腺klk是特异性的。2. nnAA-KLKs将与天然底物连接，并通过重组生产进行相应的修饰。纯化后的稳定配合物，他们是生物化学表征和结晶。如果这个过程产生适合x射线衍射的晶体，很可能就可以确定蛋白质复合物的结构。蛋白质与nnAAs的定点交联可能成为一种通用方法，适用于蛋白质与其他生物分子（如核酸、脂质和糖）的复合物。特别是在这个项目中，可以获得关于klk的三维结构元素的信息，这些信息决定了它们的底物特异性，这对于阐明生物学和疾病相关过程（例如前列腺癌）具有很高的价值。了解KLK蛋白酶的这种“三级特异性”将使制备具有前所未有功效的新药成为可能。

Many biological processes depend on proteins that bind other molecules, which are often proteins as well. Some of these complexes are very stable and can be analyzed in structural biology. However, there are also short-lived complexes, which are much more difficult to investigate. The current procedures for cross-linking molecular components of unstable complexes are rather unspecific. Thus, new approaches for site-directed linking are desirable. So-called click chemistry reactions result in the formation of covalent bonds at defined positions in proteins, among them the well-established 1,3-dipolar cycloaddition, also known as azide-alkyne click reaction, and the newly developed thiol-ene coupling for proteins. Both approaches are based on non-natural or non-canonical amino acids (nnAA), which can be incorporated into selected proteins by manipulating the genetic code and the protein synthesis in cell cultures. A suitable model system for protein-ligand interactions are the human kallikrein-related peptidases (KLKs) from prostate, in particular KLKs 2, 3, 4, 5, and 11. These serine proteases have important physiological functions in fertilization and altered roles under pathological conditions as prostate cancer.Knowledge of several KLK crystal structures is a good basis for placing nnAAs at selected positions of the proteases for linking them to other molecules. To this end, mainly inactive variants of these KLKs will be employed, which do not cleave the linked molecules after the click reactions. The two approaches of the click reactions will be applied in two steps: 1. Inactive KLK variants containing reactive nnAAs are linked with peptides containing an azide and an alkyne as nnAA reaction partner, or a cysteine and an olefinic side-chain, respectively. These peptides are derived from natural substrates, for which the prostatic KLKs are specific. 2. The nnAA-KLKs will be linked to natural substrates, with corresponding modifications by recombinant production. After purification of the stabilized complexes, they are biochemically characterized and crystallized. If this procedure yields suitable crystals for X-ray diffraction, most likely the structures of the protein complexes can be determined.Site-directed cross-linking of proteins with nnAAs could become a general method, which is applicable to protein complexes with other biomolecules, such as nucleic acids, lipids, and sugars. Especially in this project information can be gained on the three-dimensional structural elements of the KLKs, which determine their substrate specificity, which is highly valuable for elucidating biological and disease-related processes, e.g. in prostate cancer. Knowledge of this “tertiary specificity” of the KLK proteases will enable the preparation of novel drugs with unprecedented efficacy.