Homing in on the role of transition metals in the HNH motif of colicin endonucleases
Homing in on the role of transition metals in the HNH motif of colicin endonucleases
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DOI：
10.1074/jbc.274.38.27153
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发表时间：
1999-09-17
影响因子：
4.8
通讯作者：
Kleanthous, C
中科院分区：
文献类型：
--
作者：
Pommer, AJ;Kühlmann, UC;Kleanthous, C
大肠杆菌素E9的细胞毒性结构域（E9 DNA酶）是一种非特异性核酸内切酶，必须穿过两层膜才能到达其细胞靶点细菌DNA。最近的结构研究表明，大肠杆菌素DNA酶的活性位点包括在归巢核酸内切酶中发现的HNH基序，并且在该基序内结合单个过渡金属离子（Zn 2+或Ni 2+），其作用未知。在目前的工作中，我们发现，既不需要Zn 2+，也不需要Ni 2+的DNA酶的活性，而需要Mg 2+离子，但结合过渡金属的E9 DNA酶的二级和三级结构引起微妙的变化。光谱，蛋白水解和量热数据表明，伴随着1当量的Zn ~（2+），Ni ~（2+），或Co ~（2+）的结合，热力学st; Zn ~（2+）的平衡解离常数小于或等于纳摩尔，Co ~（2+）和Ni ~（2+）的平衡解离常数为微摩尔，我们的数据表明，过渡金属是大肠杆菌素DNA酶活性不是必不可少的，而是服务于结构的作用。我们推测，HNH基序已被改编为使用的核酸内切酶大肠杆菌素，因为它参与DNA识别，因为结合的金属离子的去除不稳定的DNA酶域，其易位跨细菌膜的可能先决条件。
The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn2+ or Ni2+) the role of which is unknown. In the present work we find that neither Zn2+ nor Ni2+ is required for DNase activity, which instead requires Mg2+ ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn2+, Ni2+, or Co2+, the thermodynamic st;ability of the domain increased substantially, and that the equilibrium dissociation constant for Zn2+ was less than or equal to nanomolar, while that for Co2+ and Ni2+ was micromolar, Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.