Mapping of the Signal Peptide-Binding Domain of Escherichia coli SecA Using Forster Resonance Energy Transfer
Mapping of the Signal Peptide-Binding Domain of Escherichia coli SecA Using Forster Resonance Energy Transfer
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DOI：
10.1021/bi901446r
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发表时间：
2010-02-02
期刊：
影响因子：
2.9
通讯作者：
Mukerji, Ishita
中科院分区：
文献类型：
--
作者：
Auclair, Sarah M.;Moses, Julia P.;Mukerji, Ishita
SecA ATP酶内信号肽结合结构域的鉴定是理解SecA前蛋白识别的分子基础以及阐明蛋白质易位期间该纳米颗粒的化学机械循环的重要目标。在这项研究中，福斯特共振能量转移方法被用来映射的SecA信号肽结合结构域的位置使用的功能性单胱氨酸SecA突变体和碱性磷酸酶信号肽标记适当的供体-受体荧光团的集合。荧光各向异性测量产生的平衡结合常数为1.4或10.7 μ M的碱性磷酸酶信号肽标记在残基22或2，分别与SecA，和一个信号肽结合每个SecA单体的结合化学计量。用单体偏向突变体进行的结合亲和力测量表明，信号肽与SecA单体或二聚体同样良好地结合。距离测量确定的13 SecA突变体显示，SecA信号肽结合结构域包括前蛋白交联结构域的一部分，但也包括区域的核苷酸结合结构域I，特别是螺旋支架结构域。所确定的区域位于SecA的心脏内的多结构域界面处，被对结合核苷酸、前蛋白的成熟部分和SecYEG通道重要的结构域包围并可能对其作出响应。我们的FRET映射的结合域，与通过NMR光谱鉴定的域相反，包括已被证明在易位过程中与前蛋白相互作用的双螺旋指，并且位于最近确定的SecA-SecYEG结构中的蛋白传导通道的入口处。
Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemo-mechanical cycle of this nanomotor during protein translocation. In this study, Forster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 mu M for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomer-biased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotide-binding domain I and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.