Zhang Kaiming, Horikoshi Naoki, Li Shanshan, Powers Alexander S, Hameedi Mikhail A, Pintilie Grigore D, Chae Hee-Don, Khan Yousuf A, Suomivuori Carl-Mikael, Dror Ron O, Sakamoto Kathleen M, Chiu Wah, Wakatsuki Soichi
MOE Key Laboratory for Cellular Dynamics and Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China.
Department of Bioengineering, Stanford University, Stanford, California 94305, United States.
ACS Cent Sci. 2022 Feb 23;8(2):214-222. doi: 10.1021/acscentsci.1c01090. Epub 2022 Feb 7.
Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 Å resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.
低温电子显微镜（cryo-EM）已成为开发针对人类疾病的分子疗法的一种可行的结构工具。然而，确定小于30 kDa的柔性蛋白质的结构仍然是一项挑战。CREB结合蛋白（CBP）的11 kDa KIX结构域是急性髓性白血病和其他癌症的潜在治疗靶点，该蛋白一直难以进行基于结构的抑制剂设计。在此，我们开发了一种实验方法来克服尺寸限制，通过构建蛋白质双壳结构，将KIX结构域夹在作为内壳的脱铁铁蛋白和作为外壳的麦芽糖结合蛋白之间。为了帮助目标蛋白形成均匀的取向，在目标蛋白与脱铁铁蛋白的界面处引入二硫键，从而获得了分辨率为2.6 Å的低温电子显微镜结构。我们使用分子动力学模拟来设计肽段，以阻断CBP的KIX结构域与CREB的内在无序pKID结构域之间的相互作用。双壳设计使得能够通过荧光偏振分析来确认双壳结构中的KIX结构域与这些相互作用肽段之间的结合。进一步的低温电子显微镜分析揭示了单个KIX螺旋与最佳肽段之间的螺旋-螺旋相互作用，为下一代抑制剂的开发提供了一种可能的策略。
