Molecular Biophysics Graduate Program, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Nat Commun. 2023 Apr 25;14(1):2366. doi: 10.1038/s41467-023-38031-5.
The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.
木瓜蛋白酶样蛋白酶（PLpro）是冠状病毒多功能非结构蛋白 3 的一个结构域。PLpro 可切割病毒多蛋白和与多聚泛素及保护性干扰素刺激基因 15（ISG15）结合的翻译后缀合物，后者由两个泛素样（UBL）结构域组成。尽管冠状病毒的 PLpro 序列具有保守性，但对翻译后缀合物的识别和切割仍具有不同的选择性。我们发现，SARS-CoV-2 PLpro 以纳摩尔亲和力结合人源 ISG15 和 K48 连接的二泛素（K48-Ub），并检测到其他较弱的结合模式。无束缚 PLpro 与 ISG15 和 K48-Ub 的复合物的晶体结构以及溶液 NMR 和交联质谱分析揭示了 ISG15 或 K48-Ub 的两个结构域如何在与 PLpro 的相互作用中被不同地利用。对蛋白质界面能的分析预测了两个 UBL/Ub 结构域的不同结合稳定性，实验结果验证了这一点。我们强调了如何调整底物识别以特异性切割 ISG15 或 K48-Ub 修饰物，同时保留切割单泛素缀合物的能力。这些结果突出了可抑制 PLpro 功能的替代药物靶点。
