Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.
Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.
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DOI：
10.1073/pnas.2305494120
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发表时间：
2023-09-12
影响因子：
11.1
通讯作者：
Yeates, Todd O.
中科院分区：
文献类型：
--
作者：
Castells-Graells, Roger;Meador, Kyle;Arbing, Mark A.;Sawaya, Michael R.;Gee, Morgan;Cascio, Duilio;Gleave, Emma;Debreczeni, Judit E.;Breed, Jason;Leopold, Karoline;Patel, Ankoor;Jahagirdar, Dushyant;Lyons, Bronwyn;Subramaniam, Sriram;Phillips, Chris;Yeates, Todd O.
低温电子显微镜（cryo-EM）正在成为一种主要的方法来阐明蛋白质的原子细节结构。然而，一个关键的限制是低温电镜只适用于足够大的大分子复合物。这使得许多较小尺寸的重要蛋白质，特别是那些对治疗药物开发感兴趣的蛋白质，超出了冷冻电镜的范围。我们描述了一种蛋白质工程努力，通过开发一种模块化成像支架来克服低质量限制，这种支架能够严格结合并显示几乎任何感兴趣的小蛋白质，大大增加了其有效质量。我们展示了这项技术可以用于可视化分子，例如关键的癌症蛋白，对药物设计和生物医学研究具有重要意义。冷冻电子显微镜（Cryo-EM）已经能够测定大于约50 kDa的蛋白质的结构，包括许多其他方法难以处理的蛋白质，但它在很大程度上无法测定较小的蛋白质。在这里，我们通过将小蛋白质结合到基于设计的蛋白质笼的刚性分子支架上获得小蛋白质的结构，以2.9 Å的分辨率揭示原子细节。我们将该系统应用于关键的癌症信号蛋白KRAS（大小为19 kDa），通过冷冻电镜获得了四种致癌突变变异体的结构。重要的是，与抑制剂药物（AMG510）结合的关键G12C突变体的结构与先前的晶体状态数据相比显示出显着的构象差异。这些发现突出了低温电镜支架在推进针对癌症和其他人类疾病的小治疗蛋白靶点的药物分子设计方面的前景。
Cryoelectron microscopy (cryo-EM) is emerging as a major method for elucidating the structures of proteins in atomic detail. A key limitation, however, is that cryo-EM is applicable only to sufficiently large macromolecular complexes. This places a great many important proteins of smaller size, especially those of interest for therapeutic drug development, outside the reach of cryo-EM. We describe a protein engineering effort that overcomes the lower mass limit through the development of a modular imaging scaffold able to rigidly bind and display practically any small protein of interest, greatly increasing its effective mass. We show this technology can be used to visualize molecules, such as a key cancer protein, with important implications for drug design and biomedical research. Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.
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DOI：
10.1107/s2059798318009324
发表时间：
2018-09-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
--
作者：
Afonine PV;Klaholz BP;Moriarty NW;Poon BK;Sobolev OV;Terwilliger TC;Adams PD;Urzhumtsev A
通讯作者：
Urzhumtsev A
影响因子：
64.8
作者：
Canon, Jude;Rex, Karen;Lipford, J. Russell
通讯作者：
Lipford, J. Russell
影响因子：
6.1
作者：
HENDERSON, R
通讯作者：
HENDERSON, R
影响因子：
5.5
作者：
Alford, Rebecca F.;Leaver-Fay, Andrew;Gray, Jeffrey J.
通讯作者：
Gray, Jeffrey J.
影响因子：
4.6
作者：
Coscia F;Estrozi LF;Hans F;Malet H;Noirclerc-Savoye M;Schoehn G;Petosa C
通讯作者：
Petosa C