Peptide analogues of the VanS catalytic center inhibit VanR binding to its cognate promoter.
Peptide analogues of the VanS catalytic center inhibit VanR binding to its cognate promoter.
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VanS 催化中心的肽类似物抑制 VanR 与其同源启动子的结合。
DOI：
10.1021/bi0012888
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Weisblum,B
中科院分区：
文献类型：
--
作者：
Ulijasz,AT;Kay,BK;Weisblum,B
十二聚体肽SLCHDSVIGWEC，命名为E12，是根据其能够结合VanR的能力从组合肽库中选择的，VanR是控制屎肠球菌万古霉素耐药表达的双组分信号转导反应调节因子。E12的结合定位于VanR的n端调控结构域，该结构域含有Asp-55，该残基接受活化的VanS传感器激酶中His-164的磷酸化基。E12及其相关序列SLAHDSIIGYLS，命名为E12.1，被发现抑制VanR ~ P与其同源启动子PvanH对应的DNA片段的结合。E12和E12.1都可以与VanS催化中心二聚化结构域的十八聚体序列YLAHDIKTPLTSIIGYLS（包含tyr1 -161至Ser-178）定位，这一序列通常也与VanR相互作用。对E12.1的丙氨酸取代分析发现，6个氨基酸对其抑制VanR ~ P−PvanHDNA复合物形成的能力是必不可少的。对VanS中相应氨基酸的类似分析表明，除了Leu-162→Ala和Gly-175→Ala取代干扰E12.1与蛋白质- DNA复合物形成竞争的能力外，VanS与VanR的结合能力并没有受到抑制。我们的研究结果支持了一个模型，在这个模型中，E12模仿了VanR的调控结构域与VanS的磷酸化序列相互作用，从而作为VanS的“极简”模拟物发挥作用。我们的研究结果还表明，噬菌体显示肽作为模拟相互作用蛋白质相互作用面的通用工具是有用的。
The dodecamer peptide SLCHDSVIGWEC, named E12, was selected from a combinatorial peptide library on the basis of its ability to bind to VanR, the two-component signal transduction response regulator which controls expression of vancomycin resistance inEnterococcus faecium. The binding of E12 was localized to the N-terminal, regulatory domain of VanR which contains Asp-55, the residue which accepts the phosphoryl group from His-164 in the activated VanS sensor kinase. E12, along with a related sequence SLAHDSIIGYLS, named E12.1, was found to inhibit the binding of VanR∼P to a DNA segment which corresponds to its cognate promoter PvanH. With a single gap, both E12 and E12.1 could be aligned with the octadecamer sequence YLAHDIKTPLTSIIGYLS, comprising Tyr-161 through Ser-178, of the catalytic center dimerization domain of VanS, a sequence with which VanR also normally interacts. Alanine substitution analysis of E12.1 identified six amino acids as indispensable for its ability to inhibit VanR∼P−PvanHDNA complex formation. A similar analysis of the corresponding amino acids in VanS showed a parallel dependence except for the substitutions Leu-162 → Ala and Gly-175 → Ala which interfered with the ability of E12.1 to compete with protein−DNA complex formation, but did not inhibit the ability of VanS to bind VanR. Our findings support a model in which E12 mimics the VanS phosphorylatable sequence with which the regulatory domain of VanR interacts, and thus functions as a “minimalist” analogue of VanS. Our results also indicate the usefulness of phage-displayed peptides as a general tool for mimicking the interacting faces of interacting proteins.