Wydorski Pawel M, Osipiuk Jerzy, Lanham Benjamin T, Tesar Christine, Endres Michael, Engle Elizabeth, Jedrzejczak Robert, Mullapudi Vishruth, Michalska Karolina, Fidelis Krzysztof, Fushman David, Joachimiak Andrzej, Joachimiak Lukasz A
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.
bioRxiv. 2023 Jan 19:2021.09.15.460543. doi: 10.1101/2021.09.15.460543.
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可切割病毒多聚蛋白以及与多聚泛素和保护性ISG15的翻译后缀合物，ISG15由两个泛素样（UBL）结构域组成。在冠状病毒中，尽管序列保守，但PLpro对翻译后缀合物的识别和切割表现出不同的选择性。我们发现，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）PLpro以纳摩尔亲和力结合人ISG15和K48连接的双泛素（K48-Ub₂），并检测到其他较弱的结合模式。未束缚的PLpro与ISG15和K48-Ub₂复合物的晶体结构，结合溶液核磁共振和交联质谱，揭示了ISG15或K48-Ub₂的两个结构域在与PLpro相互作用中是如何被不同利用的。蛋白质界面能量分析预测了两个UBL/Ub结构域不同的结合稳定性，并通过实验得到验证。我们强调了如何调节底物识别以特异性切割ISG15或K48-Ub₂修饰，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功能的其他可成药表面。
