Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .
Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .
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DOI：
10.1021/bi1002585
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发表时间：
2010-05-25
期刊：
影响因子：
2.9
通讯作者：
Freire, Ernesto
中科院分区：
文献类型：
--
作者：
Barrila, Jennifer;Gabelli, Sandra B.;Bacha, Usman;Amzel, L. Mario;Freire, Ernesto
冠状病毒是人类和其他哺乳动物每年呼吸道和肠道感染的很大一部分原因。这些病毒中最突出的是严重急性呼吸综合征冠状病毒（SARS-CoV），可引起人类急性呼吸道和胃肠道感染。冠状病毒的主要蛋白酶3CLpro在病毒成熟过程中起着关键作用，在冠状病毒中具有高度的结构保守性，是广谱抗病毒药物开发的关键靶点。二聚化是SARS 3CLpro功能不可缺少的条件，并通过涉及酶的直接和远程相互作用的机制进行调节。虽然二聚化界面上的许多结合相互作用已被广泛研究，但那些对远程控制很重要的相互作用尚未得到很好的理解。表征这些二聚化机制对于基于结构设计针对冠状病毒感染的新疗法非常重要。在这里，我们报道Asn28，一个位于11 Å的残基，距离最近的反向单体，对SARS 3CLpro的酶活性和二聚化至关重要。该残基对丙氨酸的突变几乎完全使酶失活，导致二聚化Kd降低19.2倍。以2.35 Å分辨率测定的N28A突变体的晶体结构揭示了Asn28在维持活性位点的结构完整性以及在二聚体界面和底物催化中参与结合的关键残基定向方面的关键作用。这些发现为SARS 3CLpro活性和二聚化的复杂调控机制提供了更深入的见解。
Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CLpro, is a key target for broad-spectrum antiviral development due to its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CLpro and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue located 11 Å away from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization Kd. The crystallographic structure of the N28A mutant determined at a 2.35 Å resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide a deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CLpro.
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DOI：
10.1016/s0140-6736(03)13077-2
发表时间：
2003-04-19
期刊：
Lancet (London, England)
影响因子：
--
作者：
Peiris JS;Lai ST;Poon LL;Guan Y;Yam LY;Lim W;Nicholls J;Yee WK;Yan WW;Cheung MT;Cheng VC;Chan KH;Tsang DN;Yung RW;Ng TK;Yuen KY;SARS study group
通讯作者：
SARS study group
DOI：
10.1016/s0140-6736(03)13967-0
发表时间：
2003-07-26
期刊：
Lancet (London, England)
影响因子：
--
作者：
Kuiken T;Fouchier RA;Schutten M;Rimmelzwaan GF;van Amerongen G;van Riel D;Laman JD;de Jong T;van Doornum G;Lim W;Ling AE;Chan PK;Tam JS;Zambon MC;Gopal R;Drosten C;van der Werf S;Escriou N;Manuguerra JC;Stöhr K;Peiris JS;Osterhaus AD
通讯作者：
Osterhaus AD
DOI：
10.1074/jbc.m310875200
发表时间：
2004-01-16
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Fan K;Wei P;Feng Q;Chen S;Huang C;Ma L;Lai B;Pei J;Liu Y;Chen J;Lai L
通讯作者：
Lai L
影响因子：
2.9
作者：
Ohtaka, H;Schön, A;Freire, E
通讯作者：
Freire, E
影响因子：
2.9
作者：
Barrila, Jennifer;Bacha, Usman;Freire, Ernesto
通讯作者：
Freire, Ernesto