The VH1 protein encoded by the highly conserved H1 locus of orthopoxviruses is a dual-specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are required for virus replication, VH1 is a prime target for the development of therapeutic inhibitors. However, the presentation of a shallow catalytic site has thwarted all drug development efforts. As an alternative to direct targeting of catalytic pockets, we describe surface contacts between VH1 and substrates that are essential for full activity and provide a new pathway for developing inhibitors of protein-protein interactions. Critical amino acid residues were manipulated by site-directed mutagenesis of VH1, and perturbation of peptide substrate interactions based on these mutations were assessed by high-throughput assays that employed surface plasmon resonance and phosphatase activities. Two positively-charged residues (Lys-20 and Lys-22) and the hydrophobic side chain of Met-60 appear to orient the polarity of the pTyr peptide on the VH1 surface, while additional amino acid residues that flank the catalytic site contribute to substrate recognition and productive dephosphorylation. We propose that the enzyme-substrate contact residues described here may serve as molecular targets for the development of inhibitors that specifically block VH1 catalytic activity and thus poxvirus replication.

中文翻译：

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牛痘VH1磷酸酶对肽的识别和去磷酸化

由正痘病毒高度保守的H1基因座编码的VH1蛋白是一种双特异性磷酸酶（DUSP），可水解病毒和宿主细胞蛋白的磷酸化酪氨酸，丝氨酸和苏氨酸残基中的磷酸基团。因为DUSP活性是病毒复制所必需的，所以VH1是开发治疗性抑制剂的主要目标。但是，浅催化位点的出现阻碍了所有药物开发的努力。作为直接靶向催化口袋的替代方法，我们描述了VH1和底物之间的表面接触，这对于充分发挥活性至关重要，并为开发蛋白质-蛋白质相互作用的抑制剂提供了新途径。关键氨基酸残基通过VH1的定点诱变来操纵，通过使用表面等离振子共振和磷酸酶活性的高通量分析评估了基于这些突变的多肽底物相互作用的干扰和干扰。Met-60的两个带正电荷的残基（Lys-20和Lys-22）和疏水性侧链似乎使VH1表面上pTyr肽的极性取向，而位于催化位点侧翼的其他氨基酸残基有助于底物识别和生产性去磷酸化。我们建议，此处描述的酶与底物的接触残基可作为开发特异性阻断VH1催化活性从而抑制痘病毒复制的抑制剂的分子靶标。Met-60的两个带正电荷的残基（Lys-20和Lys-22）和疏水性侧链似乎使VH1表面上pTyr肽的极性取向，而位于催化位点侧翼的其他氨基酸残基有助于底物识别和生产性去磷酸化。我们建议，此处描述的酶与底物的接触残基可作为开发特异性阻断VH1催化活性从而抑制痘病毒复制的抑制剂的分子靶标。Met-60的两个带正电荷的残基（Lys-20和Lys-22）和疏水性侧链似乎使VH1表面上pTyr肽的极性取向，而位于催化位点侧翼的其他氨基酸残基有助于底物识别和生产性去磷酸化。我们建议，此处描述的酶与底物的接触残基可作为开发特异性阻断VH1催化活性并由此抑制痘病毒复制的抑制剂的分子靶标。