Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.
Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.
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DOI：
10.1074/jbc.m115.651463
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发表时间：
2015-08-07
期刊：
影响因子：
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通讯作者：
Mesecar AD
中科院分区：
文献类型：
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作者：
Tomar S;Johnston ML;St John SE;Osswald HL;Nyalapatla PR;Paul LN;Ghosh AK;Denison MR;Mesecar AD
背景：3CLPro蛋白是冠状病毒多聚体加工所必需的，仅作为二聚体发挥活性。结果：MERS-CoV 3CLPro为弱结合二聚体，需要配体结合才能形成二聚体。结论：配体诱导的二聚化是MERS-CoV3CLPro在多蛋白加工过程中调节酶活性的关键机制。意义：通过配体诱导的二聚化激活可能会增加MERS-CoV 3CLPro抑制剂作为抗病毒药物的开发复杂性。所有冠状病毒，包括最近从β冠状病毒亚群中出现的中东呼吸综合征冠状病毒，在病毒复制过程中都需要NSP5蛋白水解酶(也称为3C样蛋白水解酶，3CLPro)，使其成为抗冠状病毒治疗药物开发的高价值靶点。动力学研究表明，与包括HKU4和HKU5在内的其他β冠状病毒2c成员的3CLPro相比，MERS-CoV 3CLPro处理多肽底物的效率较低，这是因为MERS-CoV 3CLPro是一种弱结合二聚体。相反，HKU4、HKU5和SARS-CoV 3CLPro酶是紧密相关的二聚体。分析超速离心研究支持MERS冠状病毒3CLPro是一种弱结合二聚体(KD∼52μm)，关闭速率较慢。合成了MERS-CoV 3CLPro的模拟多肽抑制剂，并将其用于分析性超速离心实验，结果表明MERS-CoV 3CLPro发生了明显的配体诱导的二聚反应。动力学研究还表明，由于诱导二聚反应，设计的可逆抑制剂在较低的化合物浓度下起到活化剂的作用。两个MERS-CoV 3CLPro和抑制剂复合体的初级序列比较和X射线结构分析表明，二聚体界面与HKU4-CoV和HKU5-CoV的3CLPro具有显著的结构相似性。尽管结构相似，但二聚能力的显著差异表明，远离二聚体界面的非保守氨基酸的远程相互作用可能控制MERS-CoV 3CLPro的二聚化。通过配体诱导二聚激活MERS-CoV 3CLPro似乎在2c基因组中是独一无二的，这可能会增加MERS-CoV 3CLPro抑制剂作为抗病毒药物开发的复杂性。
Background: 3CLpro protease is required for coronaviral polyprotein processing and is only active as a dimer. Results: MERS-CoV 3CLpro is a weakly associated dimer requiring ligand binding for dimer formation. Conclusion: Ligand-induced dimerization is a key mechanism for regulating the enzymatic activity of MERS-CoV 3CLpro during polyprotein processing. Significance: Activation via ligand-induced dimerization may add complexity for the development of MERS-CoV 3CLpro inhibitors as antivirals. All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CLpro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CLpro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CLpro is less efficient at processing a peptide substrate due to MERS-CoV 3CLpro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CLpro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CLpro is a weakly associated dimer (Kd ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CLpro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CLpro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CLpro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CLpro dimerization. Activation of MERS-CoV 3CLpro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CLpro inhibitors as antiviral agents.