Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease
Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease
复制标题
DOI：
10.1021/bi0490237
复制
发表时间：
2004-11-30
期刊：
影响因子：
2.9
通讯作者：
Chang, GG
中科院分区：
文献类型：
--
作者：
Chou, CY;Chang, HC;Chang, GG
SARS（严重急性呼吸综合征）是去年最严重的病毒性传染病之一，仍然是全球高度危险的公共卫生问题。探索负责其组成蛋白质分子的结构稳定性的相互作用的类型构成了找到病毒粒子的去稳定化方法的途径之一。在这项研究中，我们进行了一系列的实验来表征的二聚体冠状病毒主要蛋白酶（M-pro，3CL（pro））的四级结构。通过使用分析超滤，我们证明了二聚体SARS冠状病毒主要蛋白酶存在于溶液中的主要形式，在中性pH值的蛋白质浓度低至0.10 mg/mL。该酶开始解离在酸性和碱性pH值。离子强度对二聚体的稳定性有深远的影响，表明亚基缔合的主要作用力是离子相互作用。离子强度对蛋白酶分子的影响表现为在NaCl存在下酶的静电势轮廓的剧烈变化。晶体结构分析表明，界面离子相互作用主要是由Arg-4引起的。亚基之间的Glu-290离子对。在不同pH值下对二聚体-单体平衡的详细检查显示，Arg-4和Glu-290的表观pK（a）值分别为8.0 +/- 0.2和5.0 +/- 0.1。在这两个位置的突变降低了亚基之间的缔合亲和力，并且Glu-290突变体具有降低的酶活性。这些信息有助于寻找能够干预亚基缔合的物质，这是中和SARS冠状病毒毒力的有吸引力的靶点。
SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M-pro, 3CL(pro)). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits. Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.