Chen Qian, Zhou Junwei, Yang Zhixiang, Guo Jiahui, Liu Zimin, Sun Xinyi, Jiang Qingshi, Fang Liurong, Wang Dang, Xiao Shaobo
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China.
Comput Struct Biotechnol J. 2022 Jun 30;20:3409-3421. doi: 10.1016/j.csbj.2022.06.059. eCollection 2022.
Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CL) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CL exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CL remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CL using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CL numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CL and EAV 3CL formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CL and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CL toward P1-Gln. In turn, the T139K exchange of EAV 3CL showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CL to trigger a substrate specificity switch.
马动脉炎病毒（EAV）和猪繁殖与呼吸综合征病毒（PRRSV）是该病毒科的两个成员，对全球马和猪养殖业构成重大威胁。此前，我们和其他人证明，PRRSV 3C样蛋白酶（3CL）在P1位置（P1-Glu）具有非常高的谷氨酸（Glu）特异性。相比之下，EAV 3CL在P1位置对Glu和谷氨酰胺（Gln）均有识别。然而，动脉炎病毒3CL的P1底物特异性转变的潜在机制仍不清楚。我们使用基于环化荧光素酶的生物传感器系统地筛选了动脉炎病毒3CL的S1亚位点中的特定氨基酸，并确定Gly116、His133和Ser136（使用PRRSV 3CL编号）对于识别P1-Glu很重要，而Ser136对于识别P1-Gln不是必需的。分子动力学模拟和生化实验强调，PRRSV 3CL和EAV 3CL形成了不同的S1亚位点用于P1底物特异性转换。从机制上讲，PRRSV 3CL与底物P1-Glu（Lys138/P1-Glu）之间的特定分子间盐桥对于P1位置的高Glu特异性非常重要，并且K138T的交换（盐桥中断，从PRRSV到EAV）使PRRSV 3CL对P1-Gln的特异性发生了转变。反过来，EAV 3CL的T139K交换显示底物特异性有明显转变，使得含有P1-Glu的底物可能被更有效地识别。这些发现确定了一种进化上可行的机制，即仅通过动脉炎病毒3CL的单个突变破坏或重组盐桥来触发底物特异性转换。
