Lee Kitaik, Negi Hitendra, Chen Xiang, Atallah-Yunes Katerina, Truslow Sunny, Castelino Rithik E, Guest Mary R, Ciancone Anthony M, Tarasov Sergey G, Chari Raj, Walters Kylie J, O'Reilly Francis J
Structural System Biology Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health, Frederick, MD 21702-1201, USA.
Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health, Frederick, MD 21702-1201, USA.
bioRxiv. 2025 Aug 2:2025.07.31.667872. doi: 10.1101/2025.07.31.667872.
Proteasome function depends on a network of transient interactions that remain structurally and functionally unresolved. We developed PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry), a structural proteomics workflow that stabilizes transient interactions in cells by UV-activated crosslinking to capture topological information. Applying PhIX-MS with cryo-electron microscopy (cryo-EM), we mapped redox sensor TXNL1 at the proteasome regulatory particle (RP), placing its PITH domain above deubiquitinase RPN11 and resolving its dynamic thioredoxin domain near RPN2/PSMD1 and RPN13/ADRM1, ideally located to reduce substrates prior to proteolysis. We also resolved chaperone PSMD5 bound to RP without the proteolytic core particle (CP) where its C-terminus inserts into the ATPase pore blocking CP binding. PhIX-MS and AlphaFold modeling tether ubiquitin ligase UBE3C/Hul5 along the RP placing its catalytic site above the RPN11 active site, enabling their coupled activities. Our integrative approach enables the localization of native, low-affinity protein interactions and is broadly applicable to dynamic macromolecular assemblies.
蛋白酶体的功能依赖于一个结构和功能尚未明确的瞬时相互作用网络。我们开发了PhIX-MS（光诱导原位交联-质谱），这是一种结构蛋白质组学工作流程，通过紫外线激活交联来稳定细胞中的瞬时相互作用，以捕获拓扑信息。将PhIX-MS与冷冻电子显微镜（cryo-EM）相结合，我们在蛋白酶体调节颗粒（RP）上定位了氧化还原传感器TXNL1，将其PITH结构域置于去泛素酶RPN11上方，并在RPN2/PSMD1和RPN13/ADRM1附近解析了其动态硫氧还蛋白结构域，该位置非常适合在蛋白水解之前还原底物。我们还解析了伴侣蛋白PSMD5与RP的结合情况，此时没有蛋白水解核心颗粒（CP），其C末端插入ATP酶孔中，阻止CP结合。PhIX-MS和AlphaFold建模将泛素连接酶UBE3C/Hul5沿着RP定位，将其催化位点置于RPN11活性位点上方，从而实现它们的偶联活性。我们的综合方法能够定位天然的、低亲和力的蛋白质相互作用，并且广泛适用于动态大分子组装体。
