An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases

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.