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-Ub₂) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub₂ combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub₂ 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-Ub₂ modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

中文翻译：

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双域识别决定了 SARS-CoV-2 PLpro 对人 ISG15 和 K48 连接的双泛素的选择性

木瓜蛋白酶样蛋白酶 (PLpro) 是冠状病毒的多功能非结构蛋白 3 的一个结构域。PLpro 裂解病毒多聚蛋白和具有多聚泛素和保护性 ISG15 的翻译后缀合物，由两个泛素样 (UBL) 结构域组成。尽管序列保守，但在冠状病毒中，PLpro 对翻译后缀合物的识别和切割显示出不同的选择性。我们表明 SARS-CoV-2 PLpro 以纳摩尔亲和力结合人 ISG15 和 K48 连接的二泛素 (K48-Ub ₂ )，并检测其他较弱的结合模式。_{具有 ISG15 和 K48-Ub 2}的无束缚 PLpro 复合物的晶体结构结合溶液 NMR 和交联质谱揭示了 ISG15 或 K48-Ub _{2的两个结构域}在与 PLpro 的交互中有不同的用途。蛋白质界面能量学分析预测了实验验证的两个 UBL/Ub 结构域的不同结合稳定性。我们强调如何调整底物识别以特异性切割 ISG15 或 K48-Ub ₂修饰，同时保留切割单 Ub 缀合物的能力。这些结果突出了会抑制 PLpro 功能的替代药物表面。