Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.
Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.
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DOI：
10.1371/journal.ppat.1005045
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发表时间：
2015-07
期刊：
影响因子：
6.7
通讯作者：
Hinrichs W
中科院分区：
文献类型：
--
作者：
Zühlsdorf M;Werten S;Klupp BG;Palm GJ;Mettenleiter TC;Hinrichs W
疱疹病毒编码一种特征性丝氨酸蛋白酶，具有独特的折叠和活性位点，包括不寻常的三联体Ser-His-His。蛋白酶是病毒复制所必需的，因此构成了一个有前途的药物靶点。在溶液中，酶的无活性单体和活性二聚体形式之间存在动态平衡，据信其在蛋白水解和衣壳组装的协调中起关键调节作用。目前可用的疱疹病毒蛋白酶的晶体结构对应于二聚体状态或与改变二聚化界面的肽模拟物的复合物。相比之下，天然单体状态的结构仍然难以捉摸。在这里，我们提出了三维结构的天然单体，活性二聚体，和二异丙基氟磷酸抑制二聚体蛋白酶来自伪狂犬病病毒，猪的α疱疹病毒。这些结构，解决了通过X射线晶体学的分辨率分别为2.05，2.10和2.03 μ m，允许直接比较的主要构象状态的蛋白酶。在二聚体形式中，功能性氧阴离子孔由包含两个连续精氨酸残基（Arg 136和Arg 137）的10个氨基酸残基的环形成;两者在整个疱疹病毒中严格保守。在单体形式中，环的顶部移动约11 π，导致氧阴离子空穴的完全破坏和活性的丧失。这里描述的二聚化诱导的变构变化形成了蛋白酶的浓度依赖性激活的物理基础，这对于正确的病毒复制是必不可少的。小角X-射线散射实验证实了溶液中的单体和二聚体蛋白酶的浓度依赖性平衡。疱疹病毒编码一种独特的丝氨酸蛋白酶，这是必不可少的疱疹病毒衣壳成熟，因此是一个有趣的药物开发的目标。在溶液中，这种蛋白酶以无活性单体和活性二聚体形式的平衡存在。疱疹病毒蛋白酶的所有目前可用的晶体结构代表复合物，特别是二聚体。在这里，我们显示了第一个三维结构的天然单体形式，除了天然和化学灭活的二聚体形式的蛋白酶来源于猪疱疹病毒伪狂犬病病毒。单体和二聚体形式的比较允许预测在二聚化过程中发生的结构变化，并揭示蛋白酶活化的过程。这些新的晶体结构提供了一个合理的基础，开发药物，防止二聚化，从而阻碍疱疹病毒衣壳成熟。此外，这种机制很可能在整个疱疹病毒中是保守的。
Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution. Herpesviruses encode a unique serine protease, which is essential for herpesvirus capsid maturation and is therefore an interesting target for drug development. In solution, this protease exists in an equilibrium of an inactive monomeric and an active dimeric form. All currently available crystal structures of herpesvirus proteases represent complexes, particularly dimers. Here we show the first three-dimensional structure of the native monomeric form in addition to the native and the chemically inactivated dimeric form of the protease derived from the porcine herpesvirus pseudorabies virus. Comparison of the monomeric and dimeric form allows predictions on the structural changes that occur during dimerization and shed light onto the process of protease activation. These new crystal structures provide a rational base to develop drugs preventing dimerization and therefore impeding herpesvirus capsid maturation. Furthermore, it is likely that this mechanism is conserved throughout the herpesviruses.
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DOI：
10.1107/s090744491003982x
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Evans PR
通讯作者：
Evans PR
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
Lopez R
影响因子：
5.4
作者：
BAUM, EZ;BEBERNITZ, GA;GLUZMAN, Y
通讯作者：
GLUZMAN, Y
影响因子：
5.4
作者：
BeaudetMiller, M;Zhang, RM;Hong, Z
通讯作者：
Hong, Z