龙苷受体 n 端域的晶体结构
1Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology,Tianjin University, 2State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and Institute of Applied Ecology,Fujian Agriculture and Forestry University, 3Joint International Research Laboratory of Ecological Pest Control,Ministry of Education, Fuzhou, 4Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests,Fujian Agriculture and Forestry University
Summary
本文介绍了小菜蛾赖诺酮受体 n 端域的蛋白表达、纯化、结晶和结构测定的方案.
Abstract
开发针对昆虫赖诺丁受体 (ryr) 的有效和高效杀虫剂在农业虫害防治领域引起了极大的兴趣。迄今为止, 几种针对害虫 ryr 的二胺类杀虫剂已经商业化, 年收入达20亿美元。但由于缺乏有关昆虫 ryr 的结构信息, 对 ryr 靶向杀虫剂作用方式的理解受到限制。这反过来又限制了对害虫中杀虫剂抗药性发展的理解。钻石返回蛾 (dbm) 是一种破坏世界各地十字花科作物的破坏性害虫, 据报道, 这种害虫对菱形杀虫剂也表现出抗药性。因此, 开发针对 dbm ryr 的新型杀虫剂, 特别是针对不同于传统二胺结合位点的地区, 具有十分重要的现实意义。在这里, 我们提出了一个协议, 从结构上描述 n 终端域的 ryr 从 dbm。x 射线晶体结构是通过分子置换求解的 2.84, 该分辨率呈β-树花药折叠图案和侧翼α螺旋。该协议可用于其他领域或一般蛋白质的表达、纯化和结构表征。
Introduction
ryanodine 受体 (ryr) 是特定的离子通道, 它介导ca 2 +离子在肌肉细胞中的肉瘤-质网膜 (sr) 的渗透。因此, 它们在激励收缩耦合过程中发挥着重要作用。在其功能形式中, ryr 组装成一个同型四聚体, 其分子质量为 gt;2 mda, 每个亚基由 ~ 5000个氨基酸残基组成。在哺乳动物中, 有三种等形式: ryr1-骨骼肌类型, ryr2-心肌类型和 ryr3-在不同的组织1中普遍表达。
在昆虫中只有一种类型的 ryr, 它表现在肌肉和神经组织2中。昆虫 ryr 更类似于哺乳动物 ryr2, 序列特征约为 47%3。拜耳 (氟苯二胺)、杜邦 (氯硝酸) 和先正达 (氰基吡鱼) 等主要公司开发并销售了针对舌再产的二铵杀虫剂。自最近推出以来, 二胺类杀虫剂已成为增长最快的杀虫剂类别之一。目前, 这三种杀虫剂的年销售额已超过20亿美元, 增长率自2009年以来超过 50% (阿特拉诺娃)。
最近的研究报告了在使用这些杀虫剂4、5、6、7、8几代之后昆虫抗药性的发展。来自钻石蛾 (dbm)、 plutella xylostella ( g4946e, i4790m) 的 ryr 跨膜域的抗性突变以及番茄叶矿工tuta aicuruta的相应位置表明该地区可能与二胺杀虫剂结合, 因为该地区是众所周知的关键是门控通道 4,8,9。尽管在这一领域进行了广泛的研究, 但二胺类杀虫剂的确切分子机制仍然难以捉摸。此外, 目前尚不清楚耐药突变是直接影响与二胺的相互作用还是同种异体的相互作用。
早期的研究报告了哺乳动物种的几个 ryr 域的结构, 以及 x 射线晶体学和低温电子显微镜分别为 10、11、 12、13、14、15、16、17、18、19、20、21.但到目前为止, 还没有报道昆虫 ryr 的结构, 这使得我们无法了解受体功能的分子复杂性, 以及杀虫剂作用和杀虫剂抗药性的发展的分子机制。
在这篇手稿中, 我们提出了一个广义的协议的结构表征 n 端β-树箔域的 ryanodine 受体从钻石蛾, 一种破坏性的害虫感染世界各地的十字花科作物22。该结构是根据出版的兔子 ryr1 ntd 晶体结构23,24和低温 em 结构模型16,17,18, 19设计的。20,21. 这是为昆虫 ryr 报告的第一个高分辨率结构, 它揭示了通道门控的机制, 并为利用结构药物设计开发物种专用杀虫剂提供了一个重要模板。在结构阐明方面, 我们采用了 x 射线晶体学, 它被认为是在近原子分辨率下测定蛋白质结构的 “金标准”。虽然结晶过程是不可预测的和劳动密集型的, 这一步一步的协议将帮助研究人员表达, 纯化和表征昆虫 ryr 或任何其他蛋白质的其他领域或一般。
Protocol
1. 基因克隆、蛋白质表达和纯化 pcr 扩增与感兴趣的蛋白质相对应的 dna (dbm ryr 的残留量 1-205, genbank acc. no。afw97408) 和克隆到 pet-28a-hmt 矢量由连接独立克隆 (lic)25。该载体包含一个组氨酸标记、mbp 标记和 n-终点15处的 tev 蛋白酶裂解位点. 设计用于扩增目标基因的 lic 引物, 其与 lic 兼容的 5 ‘ 扩展:正向 lic 底漆:5 ‘ tactcacccatcatgggggggg…
Representative Results
净化 dbm ryr 的 n 端域被表示为具有六角蛋白标记、mbp 标记和 tev 蛋白酶裂解位点的融合蛋白。我们遵循五步纯化策略, 以获得一种高度纯的蛋白质, 适合结晶目的。首先, 用 ni-nta 柱 (histrap hp) 从细胞裂解液的可溶性组分中纯化了融合蛋白。其次, 对融合蛋白进行 tev 蛋白酶裂解, 用直链淀粉树脂柱去除六角蛋白 mbp-多重症, 然后采用?…
Discussion
本文描述了 dbm ryr ntd 的重组表达、纯化、结晶和结构的确定过程。对于结晶, 一个关键的要求是获得高溶解度、纯度和均匀性的蛋白质。在我们的协议中, 我们选择使用 pet-28a-hmt 矢量, 因为它包含一个六氨酸标记和 mbp 标记, 这两个标记都可用于纯化, 以获得更高的折叠纯度。此外, mbp 标记有助于目标蛋白的溶解度。我们通过连续五个步骤纯化蛋白质, 得到了高度纯净、适合结晶的蛋白质。采用自动…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究的资金由以下机构提供: 国家重点研究和发展计划 (2017yfd0201400, 2017YFD0201400)、中国国家自然科学基金 (13320103922, 31230061) 和国家基础研究项目 (973)中国 (2015cb856500, 2015cb856504)。我们感谢上海同步辐射设施 (ssrf) 波束线 bl17u1 的工作人员。
Materials
| pET-28a-HMT vector | This modified pET vector contains a hexahistidine tag, an MBP fusion protein and a TEV protease cleavage site at the N-terminus (Lobo and Van Petegem, 2009) | ||
| E. coli BL21 (DE3) strain | Novagen | 69450-3CN | |
| HisTrapHP column (5 mL) | GE Healthcare | 45-000-325 | |
| Amylose resin column | New England Biolabs | E8021S | |
| Q Sepharose high-performance column | GE Healthcare | 17-1154-01 | |
| Amicon concentrators (10 kDa MWCO) | Millipore | UFC901008 | |
| Superdex 200 26/600 gel-filtration column | GE Healthcare | 28-9893-36 | |
| Automated liquid handling robotic system | Art Robbins Instruments | Gryphon | |
| 96 Well CrystalQuick | Greiner bio-one | 82050-494 | |
| Uni-Puck | Molecular Dimensions | MD7-601 | |
| Mounted CryoLoop – 20 micron | Hampton Research | HR4-955 | |
| CryoWand | Molecular Dimensions | MD7-411 | |
| Puck dewar loading tool | Molecular Dimensions | MD7-607 | |
| Nano drop | Thermo Scientific | NanoDrop One | |
| Crystal incubator | Molecular Dimensions | MD5-605 | |
| X-Ray diffractor | Rigaku | FRX | |
| PCR machine | Eppendorf | Nexus GX2 | |
| Plasmid mini-prep kit | Qiagen | 27104 | |
| Gel extraction kit | Qiagen | 28704 | |
| SspI restriction endonuclease | NEB | R0132S | |
| T4 DNA polymerase | Novagen | 2868713 | |
| Kanamycin | Scientific Chemical | 25389940 | |
| IPTG | Genview | 367931 | |
| HEPES | Genview | 7365459 | |
| β-mercaptoethanol | Genview | 60242 | |
| Centrifuge | Thermo Scientific | Sorvall LYNX 6000 | |
| Sonnicator | Scientz | II-D | |
| Protein purification system | GE Healthcare | Akta Pure | |
| Light microscope | Nikon | SMZ745 | |
| IzIt crystal dye | Hampton Research | HR4-710 | |
| Electrophoresis unit | Bio-Rad | 1658005EDU | |
| Shaker Incubator | Zhicheng | ZWYR-D2401 | |
| Index crystal screen | Hampton Research | HR2-144 | |
| Structure crystal screen | Molecular Dimensions | MD1-01 | |
| ProPlex crystal screen | Molecular Dimensions | MD1-38 | |
| PACT premier crystal screen | Molecular Dimensions | MD1-29 | |
| JCSG-plus crystal screen | Molecular Dimensions | MD1-37 |
Riferimenti
- Giannini, G., Sorrentino, V. Molecular structure and tissue distribution of ryanodine receptors calcium channels. Medicinal Research Reviews. 15 (4), 313-323 (1995).
- Takeshima, H., et al. Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster. FEBS Letters. 337 (1), 81-87 (1994).
- Sattelle, D. B., Cordova, D., Cheek, T. R. Insect ryanodine receptors: molecular targets for novel pest control chemicals. Invertebrate Neuroscience. 8 (3), 107-119 (2008).
- Steinbach, D., et al. Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella. Insect Biochemistry and Molecular Biology. 63, 14-22 (2015).
- Wang, X., Khakame, S. K., Ye, C., Yang, Y., Wu, Y. Characterisation of field-evolved resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella, from China. Pest Management Science. 69 (5), 661-665 (2013).
- Liu, X., Wang, H. Y., Ning, Y. B., Qiao, K., Wang, K. Y. Resistance Selection and Characterization of Chlorantraniliprole Resistance in Plutella xylostella (Lepidoptera: Plutellidae). Journal of Economic Entomology. 108 (4), 1978-1985 (2015).
- Guo, L., et al. Functional analysis of a point mutation in the ryanodine receptor of Plutella xylostella (L.) associated with resistance to chlorantraniliprole. Pest Management Science. 70 (7), 1083-1089 (2014).
- Troczka, B., et al. Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor. Insect Biochemistry and Molecular Biology. 42 (11), 873-880 (2012).
- Roditakis, E., et al. Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). Insect Biochemistry and Molecular Biology. 80, 11-20 (2017).
- Borko, L., et al. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias. Acta Crystallographica Section D. 70 (Pt 11), 2897-2912 (2014).
- Sharma, P., et al. Structural determination of the phosphorylation domain of the ryanodine receptor. FEBS Journal. 279 (20), 3952-3964 (2012).
- Kimlicka, L., Lau, K., Tung, C. C., Van Petegem, F. Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface. Nature Communications. 4, 1506 (2013).
- Lau, K., Van Petegem, F. Crystal structures of wild type and disease mutant forms of the ryanodine receptor SPRY2 domain. Nature Communications. 5, 5397 (2014).
- Amador, F. J., et al. Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation "hot spot" loop. Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).
- Lobo, P. A., Van Petegem, F. Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. Structure. 17 (11), 1505-1514 (2009).
- des Georges, A., et al. Structural Basis for Gating and Activation of RyR1. Cell. 167 (1), 145-157 (2016).
- Efremov, R. G., Leitner, A., Aebersold, R., Raunser, S. Architecture and conformational switch mechanism of the ryanodine receptor. Nature. 517 (7532), 39-43 (2015).
- Peng, W., et al. Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2. Science. 354 (6310), (2016).
- Wei, R. S., et al. Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1. Cell Research. 26 (9), 977-994 (2016).
- Yan, Z., et al. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution. Nature. 517 (7532), 50-55 (2015).
- Zalk, R., et al. Structure of a mammalian ryanodine receptor. Nature. 517 (7532), 44-49 (2015).
- Furlong, M. J., Wright, D. J., Dosdall, L. M. Diamondback moth ecology and management: problems, progress, and prospects. Annual Review of Entomology. 58, 517-541 (2013).
- Amador, F. J., et al. Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation "hot spot" loop. Proceedings of the National Academy of Sciences of the United States of America. 106 (27), 11040-11044 (2009).
- Lobo, P. A., Van Petegem, F. Crystal Structures of the N-Terminal Domains of Cardiac and Skeletal Muscle Ryanodine Receptors: Insights into Disease Mutations. Structure. 17 (11), 1505-1514 (2009).
- Aslanidis, C., de Jong, P. J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Research. 18 (20), 6069-6074 (1990).
- Stepanov, S., et al. JBluIce-EPICS control system for macromolecular crystallography. Acta Crystallographica Section D. 67 (3), 176-188 (2011).
- Minor, W., Cymborowski, M., Otwinowski, Z., Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallographica Section D. 62 (Pt 8), 859-866 (2006).
- McCoy, A. J., et al. Phaser crystallographic software. Journal of Applied Crystallography. 40 (Pt 4), 658-674 (2007).
- Adams, P. D., et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D. 66 (Pt 2), 213-221 (2010).
- Zwart, P. H., Gross-Kunstleve, R. W., Adams, P. D. Xtriage and Fest: Automatic assessment of X-ray data and substructure structure factor estimation. CCP4 Newsletter. (43), 27-35 (2005).
- Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols. 10 (6), 845-858 (2015).
- Terwilliger, T. C., et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallographica Section D. 64 (Pt 1), 61-69 (2008).
- Emsley, P., Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallographica Section D. 60, 2126-2132 (2004).
- Afonine, P. V., et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallographica Section D. 68 (Pt 4), 352-367 (2012).
- Lin, L., et al. Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites. Insect Biochemistry and Molecular Biology. 92, 73-83 (2018).
- Qi, S., Casida, J. E. Species differences in chlorantraniliprole and flubendiamide insecticide binding sites in the ryanodine receptor. Pesticide Biochemistry and Physiology. 107 (3), 321-326 (2013).
Citazione di questo articolo
Nayak, B. C., Wang, J., Lin, L., He, W., You, M., Yuchi, Z. Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella. J. Vis. Exp. (141), e58568, doi:10.3791/58568 (2018).