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首页> 外文期刊>Biochemistry >Insights into Nucleotide Recognition by Cell Division Protein FtsZ from a mant-GTP Competition Assay and Molecular Dynamics
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Insights into Nucleotide Recognition by Cell Division Protein FtsZ from a mant-GTP Competition Assay and Molecular Dynamics

机译：通过mant-GTP竞争测定和分子动力学对细胞分裂蛋白FtsZ识别核苷酸的认识

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摘要

Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for newnantibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the associationninterface between monomers leads to filament disassembly.We have developed a homogeneous competitionnassay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ,nwhich detects compounds binding to the nucleotide site in FtsZmonomers andmeasures their affinities withinnthemillimolar to 10 nMrange.We have employed thismethod to determine the apparent contributions of thenguanine, ribose, and the R-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similarnrelative contributions have also been estimated through molecular dynamics and binding free energyncalculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energeticallyndominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bindnwith similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predictednaccommodation of GDP which has not been observed in the crystal structures. The binding affinities of anseries of C8-substitutedGTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlatenwith their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitorsntargeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Ournresults give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of newninhibitors, especially GTP phosphate mimetics.
机译：必需的细胞分裂蛋白FtsZ形成细菌的细胞动力学环，是新抗生素的靶标。 FtsZ单体结合GTP并组装成细丝。在单体之间的缔合界面上水解成GDP会导致细丝分解。我们已经开发出一种同质竞争分析法，利用mant-GTP与无核苷酸的FtsZ结合后的荧光各向异性变化来检测化合物与FtsZ单体中核苷酸位点的结合并测量其亲和力在毫摩尔范围内至10 nMrange。我们已采用该方法确定鸟嘌呤，核糖以及R-，β-和γ-磷酸酯对核苷酸结合自由能变化的表观贡献。通过使用FtsZ-核苷酸复合物的晶体结构的分子动力学和结合自由能计算，也估计了类似的贡献。我们发现与整个鸟苷部分相当的β-磷酸的能量主要贡献。 GTP和GDP的结合力与FtsZ单体相似。调节性γ-磷酸酯的损失导致在晶体结构中未观察到GDP的预期适应性。一系列C8取代的GTP类似物的结合亲和力，已知抑制FtsZ但不抑制真核微管蛋白装配，与它们对FtsZ聚合的抑制能力相关。我们的方法允许测试靶向其核苷酸位点的FtsZ抑制剂，以及通过虚拟筛选大型合成文库得到的化合物。我们的结果深入了解了FtsZ-核苷酸之间的相互作用，这可能在合理设计新抑制剂（尤其是GTP磷酸盐模拟物）时有用。

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