Name: production, crystallization, and structure determination of the ikk-binding domain of nemo
Uploaded: 2019-12-28T12:00:00
Description: Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

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。

1. 晶体学构造设计
<ol><li>使用T7启动子在大肠杆菌中表达的载体中克隆NEMO-EEAA序列，包括N端六核-histidine标记和蛋白酶裂解位点。 注：在此协议中，我们使用经过修改的载体，包括N端六角体-赫丁标记和烟草蚀刻病毒（TEV）裂解位10。该载体促进蛋白质结晶的He标记的裂解，在所需蛋白质序列开始之前只留下GSW残留物的短暂延伸。从中派生该的矢量和替...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20% w/v &#947;-PGA (Na+ form, LM)</td>
			<td>Molecular Dimensions</td>
			<td>MD2-100-150</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>3.5 kDa MWCO Dialysis Membrane</td>
			<td>Spectra/Por</td>
			<td>132724</td>
			<td>For dialysis removal of imidazole</td>
		</tr>
		<tr>
			<td>Amicon Stirred Cell</td>
			<td>Millipose Sigma</td>
			<td>UFSC 05001</td>
			<td>For protein concentration</td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Millipore Sigma</td>
			<td>9025-65-4</td>
			<td>For digestion of nucleic acid</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus (DE3)-RIL Competent Cells</td>
			<td>Agilent Technologies</td>
			<td>Model: 230245</td>
			<td>TEV expression</td>
		</tr>
		<tr>
			<td>CryoPro</td>
			<td>Hampton Research</td>
			<td>HR2-073</td>
			<td>Cryo-protectants kit</td>
		</tr>
		<tr>
			<td>D-Glucose (Dextrose)</td>
			<td>Millipore Sigma</td>
			<td>A9434</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Difco Terrific Broth</td>
			<td>ThermoFisher</td>
			<td>DF043817</td>
			<td>For culture growth</td>
		</tr>
		<tr>
			<td>Dithiothreitol &#62; 99%</td>
			<td>Goldbio</td>
			<td>DTT25</td>
			<td>For reduction of disulfides</td>
		</tr>
		<tr>
			<td>E. coli: Rosetta 2 (DE3)</td>
			<td>Novagen</td>
			<td>71400-3</td>
			<td>Expression of unlabeled NEMO-EEAA</td>
		</tr>
		<tr>
			<td>FORMULATOR</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Liquid handler/ screen builder</td>
		</tr>
		<tr>
			<td>HCl &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-581</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 75 pg</td>
			<td>GE Healthcare</td>
			<td>28989333</td>
			<td>For size exclusion purification</td>
		</tr>
		<tr>
			<td>HisTrap HP 5 mL column</td>
			<td>GE Healthcare</td>
			<td>17524802</td>
			<td>For purification of His-tagged NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 MIDAS</td>
			<td>Molecular Dimensions</td>
			<td>MD1-59</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 Morpheous</td>
			<td>Molecular Dimensions</td>
			<td>MD1-46</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>ThermoFisher</td>
			<td>288-32-4</td>
			<td>For elution from His-trap column</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside</td>
			<td>Goldbio</td>
			<td>I2481C5</td>
			<td>For induction of cultures</td>
		</tr>
		<tr>
			<td>MRC2 crystallization plate</td>
			<td>Hampton Research</td>
			<td>HR3-083</td>
			<td>Crystallization plate</td>
		</tr>
		<tr>
			<td>NT8 - Drop Setter</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization</td>
		</tr>
		<tr>
			<td>pET-16b</td>
			<td>Millipore Sigma</td>
			<td>69662</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pET-45b</td>
			<td>Millipore Sigma</td>
			<td>71327</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>ThermoFisher</td>
			<td>36978</td>
			<td>For inhibition of proteases</td>
		</tr>
		<tr>
			<td>Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti</td>
			<td>Beckmann Coulter</td>
			<td>339160</td>
			<td>Ultracentrifuge bottle</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 20,000</td>
			<td>Hampton Research</td>
			<td>HR2-609</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pRK793 (TEV)</td>
			<td>Addgene</td>
			<td>Plasmid 8827</td>
			<td>For TEV production</td>
		</tr>
		<tr>
			<td>QuikChange XL II</td>
			<td>Agilent Technologies</td>
			<td>200522</td>
			<td>Site directed mutagenesis</td>
		</tr>
		<tr>
			<td>Required Cap Assembly:</td>
			<td>Beckmann Coulter</td>
			<td>355623</td>
			<td>Ultracenttrifuge bottle cap</td>
		</tr>
		<tr>
			<td>ROCK IMAGER</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization Imager</td>
		</tr>
		<tr>
			<td>Seed Bead Kit</td>
			<td>Hampton Research</td>
			<td>HR2-320</td>
			<td>Seed generation</td>
		</tr>
		<tr>
			<td>Sodium Chloride &#8805; 99%</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)</td>
			<td>Goldbio</td>
			<td>TCEP1</td>
			<td>Reducing agent</td>
		</tr>
		<tr>
			<td>The Berkeley Screen</td>
			<td>Rigaku</td>
			<td>MD15-Berekely</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>The PGA Screen</td>
			<td>Molecular Dimensions</td>
			<td>MD1-50</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Tris &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-589</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Ultrapure Tris Buffer (powder format)</td>
			<td>Thermofisher</td>
			<td>15504020</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher</td>
			<td>29700</td>
			<td>For denaturation of NEMO-EEAA</td>
		</tr>
	</tbody>
</table>

production, crystallization, and structure determination of the ikk-binding domain of nemo

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。

NEMO IKK结合域的克隆、表达和纯化。 本研究中遵循的协议获得NEMO-EEAA的最终序列（图1A），它产生衍射质量晶体，涉及所有中间结构的表达和表征，包括在N和或C-终点站添加线圈适配器，突变C76A，C95S和突变E56A，E57A。图1B显示了在整个纯化过程中收集的样品的SDS-PAGE凝胶，如图1C</strong...

Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

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。

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

该协议有助于成功确定 NEMO 的 IKK 绑定域，其无边界形式。及其生产和结晶的细节。该协议，利用这种稳定，通过盘绕线圈适配器对NEMO的 IKK 绑定域片段的本机确认，从而促进结晶和结构确定。作为靶向，NEMO的结构生物学为治疗炎症和自身免疫性疾病和癌症的NEMO抑制剂的发展提供了重要优势。该协议可以扩展到NEMO复合物的结构测定，与肽或小分子抑制剂的药物发现或优化。在蛋白质生产阶段，蛋白质的再折叠和浓度对于获得纯NEMO二丁剂至关重要。限制聚合并确保结晶条件下的溶解性。演示程序将是塔玛和艾米，研究生在我们的实验室。首先添加20毫升的很棒的肉汤溶液。和20微升100毫克每毫升库存溶液的安西林。到 125 毫升埃伦迈尔烧瓶。其次是BL21DE3称能力细胞的一些微升细胞甘油储存，从80摄氏度的储存。使用矢量转换。在一夜之间摇动启动培养体后，以每分钟37摄氏度和220次旋转的速度，将细胞稀释为0.1和250毫升的OD600和250毫升的特好肉汤。将安西林加入每毫升100微克的最终浓度。当培养物的OD600达到0.8比1时，将IPTG添加到培养物中，加入500微摩尔浓度。在37摄氏度下生长细胞4小时，直到OD600达到6到10。在孵化结束时，通过离心沉淀细胞。并在40毫升的解液缓冲液中重新悬浮颗粒。将重新悬浮的细胞分成两个20至25毫升的等分。使用法国印刷机在寒冷的房间里对每平方英寸的压力施加大约25，000磅的压力，每分两位到三倍。接下来，将尿素加入细胞落酸，最终浓度为八摩尔。在摇摇平台上孵育细胞溶液2至16小时，在室温下。第二天早上，将落酸转移到超离心管，每个管至少满四分之三。和乌尔塔离心机的莱酸盐，45分钟。将超自然剂放入50毫升锥形管中。将尿素孵育的中分液加载到 IMAC 柱上，每分钟三毫升。当所有上流板都运行到列中时，以每分钟 3 毫升的速度用绑定缓冲区将列洗涤 10 列卷。并在 12 列体积梯度上对 NEMO-EEAA 蛋白进行 10 至 500 毫摩尔的梯度洗脱。收集一毫升的分水，放入一个分数收集板。SDS页面分析后，提取含有纯靶蛋白的分数，根据标准方案通过布拉德福德测定测量蛋白质浓度。选择含有蛋白质的逃避分数。要切开 HIS6 标签并从样品中去除多余的 imidazole，请将 TEV 蛋白酶（重量比为 1 至 10）添加到目标蛋白样品中。将样品在四升20毫摩尔三叶草中隔夜膨胀。150毫摩尔氯化钠和两毫摩尔DTT溶液。第二天早上，将 TEV 切割的 NEMO-EEAA 样品加载到第二个 IMAC 柱上，每分钟一毫升。在 96 碗分数收集板中收集流在一毫升分数中。用五卷 20 毫摩尔三升、150 毫摩尔氯化钠和 10 毫摩尔 imidazole 溶液清洗柱，每分钟一毫升。上次洗涤后，将 TEV 和未洁的 HIS6 NEMO-EEAA，用三列卷 20 毫摩尔三叶、150 毫摩尔氯化钠、500 毫摩尔伊米达佐尔和两毫摩尔 DTT 溶液，放入 50 毫升烧瓶中。将流拉过馏分，包含切割的NEMO-EEAA构造，用搅拌细胞集中器将样品浓缩到5毫升。过夜透析后，将样品的5毫升加载到16毫米60厘米大小的排除色谱柱上。根据需要根据样品体积重复。每分钟一毫升，在两毫摩尔三叶草，100毫摩尔氯化钠和两毫摩尔滴滴涕溶液。拉出与一毛钱 NEMO-EEAA 对应的分数后，将样品浓缩在搅拌细胞集中器中，分子量切断膜三千吨，最终浓度为 113 微摩尔。然后，将蛋白质储存在摄氏四度，长达一个月。对于稀疏基质筛选，在两滴室结晶板的 96 孔中，每个孔中加入 60 微升稀疏基质溶液。用于坐落蒸汽扩散。并将蛋白质溶液添加到机器人滴液中。接下来，使用机器人滴液器，以每毫升1.65毫克的速度分配100纳米的蛋白质溶液，以一比一的比例分配储液溶液，最终体积为200纳米升。并添加66纳米升的蛋白质溶液与134纳米升的储层溶液，最终体积200纳米升滴2。然后，立即用三英寸宽的密封胶带密封板。然后，将托盘存放在结晶成像器存储中，温度为 20 摄氏度。检查自动收集的图像，是否存在晶体，从存储板后两天开始。对于种子库存的生成，从种子生成套件中，将含有感兴趣的晶体的整个滴转移到 50 微升的结晶条件溶液中。和漩涡种子库存，20秒的脉动和10秒的休息，三分钟。然后，以1到10个增量连续稀释种子库存，至1至10，000。并在四摄氏度下储存稀释液，直到进一步使用。一到两天前装运到同步加速器，从井顶部切割胶带，与感兴趣的晶体。并添加0.5微升的结晶溶液，含有12%一、二丙烷DIO低温保护剂，直接到井中。用低温循环轻轻将晶体分离。将晶体从井中循环，将含有低温环的晶体存放在浸于液氮中的冰球中，直到装运进行X射线衍射。接收 X 射线衍射数据后，处理 STAR 和 ISO 服务器中的缩放强度。使用 X 射线晶体学索引切断均值，数据衍射极限表面为 1.2。并加载到凤凰城。要确定结构，请使用GCN4的X射线结构作为分子替换的搜索模型，使用凤凰城的MRage。4DMD 结构，将定义为一个整体。MRage 解决方案将成功地构建到结构部分，对应于 NEMO-EEAA 的端端子线圈线圈适配器。搜索模型，用于两个链条的暗淡。在第一个IMAC柱纯化之前，过度表达的蛋白质出现在一个带子上，大约14千吨分子量标记。并在第一次洗脱后，分别以14公斤和28千吨显示单体带和二丁带。TEV 裂解实际上是完整的，在洗脱之后，通过第二个 IMAC 列。几乎完全作为一个暗淡，在预期的分子量。大小排除色谱，显示单个峰值，从 60 到 65 毫升之间，谁是对暗光的反应。精细筛选产生的晶体可用于生产用于生产NEMO-EEAA晶体的种子库存，用于数据收集。在这里，显示了具有代表性的NEMO-EEAA晶体衍射剖面图。对NEMO-EEAA蛋白的结构分析揭示了一个同质的不规则平行盘绕，长度约为175个焦虑。常规盘绕区域，包括理想的线圈线圈适配器序列，在终点。和 NEMO 正确序列的前两个七氯图。一个常规的线圈，也出现在C术语。从 NEMO 残留物 97 开始，包括 C 端子的理想线圈线圈适配器。IKK beta 绑定结构，显示更开放的线圈确认，以适应配体。与更大的间距间距 1 到 2.2 焦虑在这个区域。NEMO中配体的结构，通过计算方法为抑制剂的设计提供了一个新的目标。和与小分子库结合口袋的视觉筛选。我们现在有一条在具有小分子配体的复杂中实现工程性NEMO构造结晶的路径。帮助开发和优化新型抑制剂。

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