Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
J Struct Biol. 2023 Dec;215(4):108022. doi: 10.1016/j.jsb.2023.108022. Epub 2023 Aug 30.
Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
被膜蛋白是能够选择性地包裹特定货物酶的自组装蛋白纳米容器。被膜蛋白广泛存在于细菌和古菌门中，参与氧化应激抵抗、铁储存和硫代谢。被膜蛋白壳表现出二十面体几何形状，由 60、180 或 240 个相同的蛋白质亚基组成。货物的封装是通过靶向肽或结构域与被膜蛋白壳的内部表面的特异性相互作用介导的，这些靶向肽或结构域存在于所有货物蛋白中，并且在壳自组装过程中发生。在这里，我们报告了来自粘球菌的异源生产和高载货物的 T3 被膜蛋白壳的 2.53Å 冷冻电镜结构，并探索了该系统的结构异质性。我们发现，极高的货物负载会导致大量变形和异常的壳形成，这可能是由于货物蛋白的不利空间位阻和壳构象变化的组合造成的。基于我们的冷冻电镜结构，我们确定并分析了靶向肽-壳结合模式。我们发现离子和疏水相互作用都介导靶向肽的结合。我们的结果将指导未来为生物医学和生物技术应用而进行的理性工程被膜蛋白的尝试。
