Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
UC Berkeley-UCSF Joint Graduate Program in Bioengineering, University of California, San Francisco, CA 94143.
Proc Natl Acad Sci U S A. 2022 Aug 16;119(33):e2207200119. doi: 10.1073/pnas.2207200119. Epub 2022 Jul 19.
The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.
能够生成折叠和有功能的蛋白质是结构生物学和许多其他生物学科学的必要条件。对于人类细胞中许多重要的生物医学靶点，包括膜蛋白和大型大分子组装体，这一任务尤其具有挑战性，阻碍了对其机制的研究和药物开发工作。在这里，我们描述了一种结合 CRISPR-Cas 基因编辑和荧光激活细胞分选的方法，用于快速标记和纯化 HEK 细胞中的内源性蛋白质，以进行结构表征。我们应用这种方法来研究来自 HEK 细胞的人蛋白酶体，并迅速确定了主要蛋白酶体复合物的低温电子显微镜结构，包括完整的人 PA28αβ-20S 的高分辨率结构。我们的结构揭示了具有 3α/4β 亚基组成的 PA28 与 20S 蛋白酶体紧密结合。添加亲水性肽表明，通过 PA28 进入的多肽在进入蛋白酶体的降解腔之前被保持在 20S 的前室中。这项研究为重要的蛋白酶体复合物提供了关键的见解，并展示了从内源性来源标记蛋白质的关键方法。
