Characterization of the zinc binding activity of the rubella virus nonstructural protease
Characterization of the zinc binding activity of the rubella virus nonstructural protease
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DOI：
10.1128/jvi.74.13.5949-5956.2000
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发表时间：
2000-07-01
影响因子：
5.4
通讯作者：
Frey, TK
中科院分区：
文献类型：
--
作者：
Liu, X;Yang, J;Frey, TK
风疹病毒（rubella virus，RUB）非结构（nonstructural，NS）蛋白（nonstructural protein，NSP）ORF编码蛋白酶，该蛋白酶在单个位点切割NSP前体（240 kDa）以产生两种产物。将切割位点突变引入到RUB感染性cDNA克隆中并发现是致命的，表明NSP前体的切割对于RUB复制是必需的。基于计算机比对，预测RUB NS蛋白酶是具有残基Cys 1152和His 1273作为催化二联体的木瓜蛋白酶样半胱氨酸蛋白酶（PCP）;然而，最近发现RUB NS蛋白酶需要二价阳离子如Zn、Co和Cd来获得活性（X，Liu，S. L，Ropp，R，J，杰克逊，and T，K，Frey，J，Virol，72：4463-1466，1998）。为了分析金属阳离子结合在蛋白酶活性中的功能，使用NSP ORF内的最小NS蛋白酶结构域进行Zn结合研究。当由细菌表达为麦芽糖结合蛋白（MBP）融合蛋白时，NS蛋白酶在Zn存在下变性和重折叠时在细菌中和纯化后在体外均表现出活性。原子吸收分析检测到以这种方式重折叠的每摩尔蛋白质结合1.6摩尔Zn。单个结构域内的MBP融合蛋白酶的表达和分析，通过Zn-65结合试验揭示了两个Zn结合域：一个位于一个预测的金属结合基序开始于Cys 1175和其他一个接近切割位点。突变研究表明，第一个结构域中的Cys 1175和Cys 1178以及第二个结构域中的Cys 1227和His 1273，即预测的催化位点中的His是锌结合所必需的，所有这些残基也是蛋白酶活性所必需的，其他几个不参与锌结合的Cys残基也是如此。对MBP-NS蛋白酶融合蛋白的远紫外圆二色性（CD）分析表明，该蛋白酶结构域含有大量的α-螺旋结构，这与二级结构预测的结果一致。远紫外-CD和荧光研究都表明，锌对融合蛋白的整体结构没有产生主要影响。最后，蛋白酶抑制剂测定发现，蛋白酶活性可以被金属离子螯合剂和金属蛋白酶抑制剂卡托普利阻断。结合先前预测的催化位点His 1273对锌结合是必需的这一发现，这表明RUBNS蛋白酶实际上是一种新的病毒金属蛋白酶而不是PCP。
The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products, A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X, Liu, S. L, Ropp, R, J, Jackson, and T, K, Frey, J, Virol, 72:4463-1466, 1998), To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF, When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn, Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn-65 binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding, All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV-CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein, Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril, In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.