PMID- 35832618 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20220716 IS - 2001-0370 (Print) IS - 2001-0370 (Electronic) IS - 2001-0370 (Linking) VI - 20 DP - 2022 TI - An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases. PG - 3409-3421 LID - 10.1016/j.csbj.2022.06.059 [doi] AB - Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae 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(pro)) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CL(pro) 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(pro) remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CL(pro) using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CL(pro) 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(pro) and EAV 3CL(pro) formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CL(pro) 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(pro) toward P1-Gln. In turn, the T139K exchange of EAV 3CL(pro) 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(pro) to trigger a substrate specificity switch. CI - (c) 2022 The Author(s). FAU - Chen, Qian AU - Chen Q AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Zhou, Junwei AU - Zhou J AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Yang, Zhixiang AU - Yang Z AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Guo, Jiahui AU - Guo J AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Liu, Zimin AU - Liu Z AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Sun, Xinyi AU - Sun X AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Jiang, Qingshi AU - Jiang Q AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Fang, Liurong AU - Fang L AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Wang, Dang AU - Wang D AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. FAU - Xiao, Shaobo AU - Xiao S AD - State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. AD - Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China. LA - eng PT - Journal Article DEP - 20220630 PL - Netherlands TA - Comput Struct Biotechnol J JT - Computational and structural biotechnology journal JID - 101585369 PMC - PMC9271976 OTO - NOTNLM OT - 3C-like protease OT - Arterivirus OT - Molecular dynamics simulation OT - Salt bridge OT - Site-directed mutagenesis OT - Substrate specificity switch COIS- The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2022/07/15 06:00 MHDA- 2022/07/15 06:01 PMCR- 2022/06/30 CRDT- 2022/07/14 02:26 PHST- 2022/02/21 00:00 [received] PHST- 2022/06/26 00:00 [revised] PHST- 2022/06/27 00:00 [accepted] PHST- 2022/07/14 02:26 [entrez] PHST- 2022/07/15 06:00 [pubmed] PHST- 2022/07/15 06:01 [medline] PHST- 2022/06/30 00:00 [pmc-release] AID - S2001-0370(22)00273-2 [pii] AID - 10.1016/j.csbj.2022.06.059 [doi] PST - epublish SO - Comput Struct Biotechnol J. 2022 Jun 30;20:3409-3421. doi: 10.1016/j.csbj.2022.06.059. eCollection 2022.