NEMO IKK绑定域的生产、结晶和结构确定
Summary
我们描述了通过X射线晶体学测定NEMOIKK结合域的结构确定方案。这些方法包括蛋白质表达、纯化和表征，以及成功优化晶体和确定无结合形式蛋白质的结构的策略。
Abstract
NEMO 是一种脚手架蛋白，通过将 IKK 复合物与激酶 IKK® 和 IKK® 组装在 NF-+B 通路中起着至关重要的作用。激活后，IKK复合磷酸化I+B分子，导致NF-βB核易位和目标基因的激活。抑制NEMO/IKK相互作用是NF-βB通路活性调节的一种有吸引力的治疗范例，使NEMO成为抑制剂设计和发现的目标。为了便于NEMO抑制剂的发现和优化过程，我们设计了NEMO的IKK结合域的改进结构，允许以apo形式和与小分子量抑制剂结合的蛋白质的结构测定。在这里，我们介绍了用于NEMOIKK绑定域的设计、表达和结构特征化的策略。蛋白质在大肠杆菌细胞中表达，在变性条件下溶解，并通过三个色谱步骤进行纯化。我们讨论了用于结构测定的晶体获取协议，并描述了数据采集和分析策略。这些方案将广泛适用于NEMO和小分子抑制剂复合物的结构测定。
Introduction
NF-βB通路被激活，以响应各种刺激，包括细胞因子，微生物产品和压力，以调节目标基因的表达负责炎症和免疫反应，细胞死亡或生存和增殖1。包括炎症和自身免疫性疾病以及癌症2、3、4、5在内的病理学都与途径的过度激活有关，这使得NF-βB活性的调节成为开发新疗法6、7的首要目标。
规范的NF-βB通路与负责淋巴组织生成和B细胞活化的非规范通路有区别，前者依赖脚手架蛋白NEMO（NF-B必需调制器8），以组装IKK复合物与激酶IKK®和IKK®。IKK复合物负责I+B+（βB抑制剂）的磷酸化，以降解为目标，释放NF-βB二聚体，将NF-βB二聚体转移到核进行基因转录1，因此是开发调节NF-B活性抑制剂的诱人靶点。
我们的研究重点是NEMO和IKK+之间的蛋白质-蛋白质相互作用的表征，针对NEMO开发IKK复杂形成小分子抑制剂。NEMO的最小结合域，需要结合IKK®，包括残留物44-111，其结构已确定在复杂的与对应于IKK®序列701-7459的肽。NEMO 和 IKK® 形成四螺旋束，其中 NEMO 二分器将 IKK® （701-745） 的两个螺旋体容纳在具有扩展交互接口的拉长开槽中。IKK®（734-742），也称为NEMO绑定域（NBD），定义了最重要的结合热点，其中两个基本色氨酸（739，741）深埋在NEMO口袋中。复杂结构的细节有助于针对NEMO的小分子抑制剂的基于结构的设计和优化。同时，很难在NEMO中重现由在晶体中观察到的长IKK®（701-745）的基基（701-745）的结合引起的整个构象变化（即NEMO盘绕-螺旋二聚体）的完全构象变化，而未结合的NEMO或NEMO与小分子抑制剂结合的结构可能代表了基于结构的药物设计和抑制剂优化的更好目标。
全长NEMO和包含IKK绑定域的较小截断构造已经证明，通过X射线晶体学和核磁共振（NMR）方法10，在未绑定形式的结构确定中，我们设计了一个改进版本的NEMO的IKK绑定域。事实上，未绑定形式的NEMO（44-111）只是部分折叠，并经历构象交换，因此，我们设置稳定其二体结构，盘绕折叠和稳定，同时保持IKK®的绑定亲和力。通过在蛋白质的N-和C-termini上附加理想二分线圈序列11的三个七分体，以及一系列四点突变，我们产生了NEMO-EEAA，一个构造完全二分体并折叠在一个盘绕线圈中，从而挽救了IKK结合亲和力到全长NEMO12的纳米摩尔范围。作为另一个优势，我们希望盘绕式适配器（基于GCN4序列）将促进结晶，并最终通过分子替换帮助X射线结构确定。卷绕式适配器也同样被利用，既提高了稳定性，改善了溶液行为，又有利于三角线圈和抗体片段13、14的结晶。NEMO-EEAA 易于表达和纯化从Escherichia. 大肠杆菌细胞与可夹层的Histidine标签，是可溶性的，折叠在一个稳定的二分卷线圈，并易于结晶，与衍射到1.9°。GCN4有序盘绕线圈区域的存在，可进一步帮助使用GCN415的已知结构进行分子替换，从而逐步利用NEMO-EEAA晶体的数据。
鉴于使用 apo-NEMO-EEAA 获得的结果，我们相信此处描述的协议也可以应用于存在小肽（如 NBD 肽）或小分子抑制剂的 NEMO-EEAA 的结晶，目的是了解 NEMO 抑制的要求以及初始铅抑制剂对高亲和力的基于结构的优化。鉴于许多盘绕域的可塑性和动态性16，使用盘绕式适配器在帮助结构确定方面可以发现更普遍的适用性。
Protocol
Representative Results
Discussion
NEMO在未绑定形式的结晶尝试没有成功，包括使用全长蛋白质的尝试和包括IKK结合域的几种截断结构。我们对NEMO的IKK结合域（残留物44-111）的生物物理表征，通过循环双色、NMR光谱和荧光各向异性表明，该结构，尽管能够结合IKK®，但存在于一种构象交换状态，不适合结晶9，10。我们的方法涉及设计 NEMO 的 IKK 绑定域，该域采用更稳定、折叠和原生样的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢D.Madden教授在整个项目中进行了许多有益的讨论。我们感谢D.Bolon教授赠送含有优化的GCN4线圈的质粒。我们感谢郭博士的NEMO质粒。我们感谢克里斯蒂娜·阿诺迪、塔马尔·巴西亚什维利和艾米·肯尼迪演示了这个程序。我们感谢达特茅斯生物晶体学核心设施以及化学、生物化学和细胞生物学系对晶体成像设备的使用，并感谢 BioMT 人员的支持。这项研究使用了国家同步辐射光源II的AMX光束线，这是美国能源部（DOE）科学用户设施办公室，由布鲁克黑文国家实验室根据合同号为能源能源科学办公室运营。DE-SC0012704。我们感谢NSLS II的工作人员的支持。这项工作由NIH赠款R03AR066130、R01GM133844、R35GM128663和P20GM1131332以及Munck-Pfefferkorn小说和互动赠款资助。
Materials
| 20% w/v γ-PGA (Na+ form, LM) | Molecular Dimensions | MD2-100-150 | For fine screen crystallization of NEMO-EEAA |
| 3.5 kDa MWCO Dialysis Membrane | Spectra/Por | 132724 | For dialysis removal of imidazole |
| Amicon Stirred Cell | Millipose Sigma | UFSC 05001 | For protein concentration |
| Ammonium Chloride | Millipore Sigma | G8270 | For minimal media labeling |
| Benzonase Nuclease | Millipore Sigma | 9025-65-4 | For digestion of nucleic acid |
| BL21-CodonPlus (DE3)-RIL Competent Cells | Agilent Technologies | Model: 230245 | TEV expression |
| CryoPro | Hampton Research | HR2-073 | Cryo-protectants kit |
| D-Glucose (Dextrose) | Millipore Sigma | A9434 | For minimal media labeling |
| Difco Terrific Broth | ThermoFisher | DF043817 | For culture growth |
| Dithiothreitol > 99% | Goldbio | DTT25 | For reduction of disulfides |
| E. coli: Rosetta 2 (DE3) | Novagen | 71400-3 | Expression of unlabeled NEMO-EEAA |
| FORMULATOR | Formulatrix | Liquid handler/ screen builder | |
| HCl – 1.0 M Solution | Hampton Research | HR2-581 | For fine screen crystallization of NEMO-EEAA |
| HiLoad 16/600 Superdex 75 pg | GE Healthcare | 28989333 | For size exclusion purification |
| HisTrap HP 5 mL column | GE Healthcare | 17524802 | For purification of His-tagged NEMO-EEAA |
| HT 96 MIDAS | Molecular Dimensions | MD1-59 | For sparse matrix screening of NEMO-EEAA |
| HT 96 Morpheous | Molecular Dimensions | MD1-46 | For sparse matrix screening of NEMO-EEAA |
| Imidazole | ThermoFisher | 288-32-4 | For elution from His-trap column |
| Isopropyl-beta-D-thiogalactoside | Goldbio | I2481C5 | For induction of cultures |
| MRC2 crystallization plate | Hampton Research | HR3-083 | Crystallization plate |
| NT8 – Drop Setter | Formulatrix | Crystallization | |
| pET-16b | Millipore Sigma | 69662 | For cloning of NEMO-EEAA |
| pET-45b | Millipore Sigma | 71327 | For cloning of NEMO-EEAA |
| Phenylmethylsulfonyl fluoride | ThermoFisher | 36978 | For inhibition of proteases |
| Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti | Beckmann Coulter | 339160 | Ultracentrifuge bottle |
| Polyethylene Glycol 20,000 | Hampton Research | HR2-609 | For fine screen crystallization of NEMO-EEAA |
| pRK793 (TEV) | Addgene | Plasmid 8827 | For TEV production |
| QuikChange XL II | Agilent Technologies | 200522 | Site directed mutagenesis |
| Required Cap Assembly: | Beckmann Coulter | 355623 | Ultracenttrifuge bottle cap |
| ROCK IMAGER | Formulatrix | Crystallization Imager | |
| Seed Bead Kit | Hampton Research | HR2-320 | Seed generation |
| Sodium Chloride ≥ 99% | Millipore Sigma | S9888 | For buffering of purification solutions |
| TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride) | Goldbio | TCEP1 | Reducing agent |
| The Berkeley Screen | Rigaku | MD15-Berekely | For sparse matrix screening of NEMO-EEAA |
| The PGA Screen | Molecular Dimensions | MD1-50 | For fine screen crystallization of NEMO-EEAA |
| Tris – 1.0 M Solution | Hampton Research | HR2-589 | For fine screen crystallization of NEMO-EEAA |
| Ultrapure Tris Buffer (powder format) | Thermofisher | 15504020 | For buffering of purification solutions |
| Urea | ThermoFisher | 29700 | For denaturation of NEMO-EEAA |
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