von Rosen Tatjana, Zdanowicz Rafal, El Hadeg Yasser, Afanasyev Pavel, Boehringer Daniel, Leitner Alexander, Glockshuber Rudi, Weber-Ban Eilika
Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.
International Institute of Molecular Mechanisms and Machines, Polish Academy of Sciences, Warsaw, Poland.
Nat Commun. 2025 Mar 28;16(1):3042. doi: 10.1038/s41467-025-58073-1.
Mycobacteria harbor a proteasome that was acquired by Actinobacteria through horizontal gene transfer and that supports the persistence of the human pathogen Mycobacterium tuberculosis within host macrophages. The core particle of the proteasome (20S CP) associates with ring-shaped activator complexes to degrade protein substrates. One of these is the bacterial proteasome activator Bpa that stimulates the ATP-independent proteasomal degradation of the heat shock repressor HspR. In this study, we determine the cryogenic electron microscopy 3D reconstruction of the complex between Bpa and its natural substrate HspR at 4.1 Å global resolution. The resulting maps allow us to identify regions of Bpa that interact with HspR. Using structure-guided site-directed mutagenesis and in vitro biochemical assays, we confirm the importance of the identified residues for Bpa-mediated substrate recruitment and subsequent proteasomal degradation. Additionally, we show that the dodecameric Bpa ring associates asymmetrically with the heptameric α-rings of the 20S CP, adopting a conformation resembling a hinged lid, while still engaging all seven docking sites on the proteasome.
分枝杆菌含有一种蛋白酶体，该蛋白酶体是放线菌通过水平基因转移获得的，支持人类病原体结核分枝杆菌在宿主巨噬细胞内的存活。蛋白酶体的核心颗粒（20S CP）与环形激活复合物结合以降解蛋白质底物。其中之一是细菌蛋白酶体激活剂Bpa，它刺激热休克阻遏物HspR的不依赖ATP的蛋白酶体降解。在本研究中，我们以4.1Å的整体分辨率确定了Bpa与其天然底物HspR之间复合物的低温电子显微镜3D重建。所得图谱使我们能够识别Bpa与HspR相互作用的区域。使用结构导向的定点诱变和体外生化分析，我们证实了所鉴定的残基对于Bpa介导的底物募集和随后的蛋白酶体降解的重要性。此外，我们表明，十二聚体Bpa环与20S CP的七聚体α环不对称结合，采用类似于铰链盖的构象，同时仍与蛋白酶体上的所有七个对接位点结合。
