Toward ligand identification within a CCHHC zinc-binding domain from the NZF/MyT1 family
Toward ligand identification within a CCHHC zinc-binding domain from the NZF/MyT1 family
复制标题
DOI：
10.1021/ic990913y
复制
发表时间：
2000-01-24
影响因子：
4.6
通讯作者：
Berg, JM
中科院分区：
文献类型：
--
作者：
Blasie, CA;Berg, JM
最近已经鉴定了一个蛋白质家族，其包含具有共有序列Cys-X-4-Cys-X-4-His-X-7-His-X-5-Cys的假定的锌结合结构域，但是尚未研究这些结构域的金属结合和结构特性。这一共识是惊人的，因为存在五个保守的潜在的锌结合残基。已经合成并表征了对应于来自转录因子NZF-1（下文称为NZF-13）的第三假定锌结合结构域的肽。该肽的钴（II）络合物的W-可见吸收光谱特性表明，金属结合是四面体的，并且可见吸收带的位置表明由三个半胱氨酸和一个组氨酸的协调。为了鉴定两个保守的组氨酸残基中的哪一个充当金属结合残基，还合成了两个组氨酸至丙氨酸的变体肽。这两种变体肽结合钴（II）在一个四面体的方式;更换的第一个两个组氨酸的吸收光谱特征的详细形状上有一个更大的影响比更换的第二个组氨酸。这些结果表明金属配位残基（斜体）是Cys-X-4-Cys-X-4-His-X-7-His-X-5-Cys。然而，用丙氨酸同时取代两个组氨酸残基会产生一种对金属结合特性影响更大的肽。这些观察结果表明，在单取代肽中观察到的相对温和的影响可能是由于涉及剩余组氨酸的金属相互作用。由于这些现象，将需要进一步的研究，以建立更明确的两个组氨酸残基在金属结合的作用和潜在的意义，明显的替代组氨酸配位。
A family of proteins that contain presumed zinc-binding domains with the consensus sequence Cys-X-4-Cys-X-4-His-X-7-His-X-5-Cys has recently been identified, but the metal binding and structural properties of these domains have not been investigated. This consensus is striking because of the presence of five conserved potential zinc-binding residues. A peptide corresponding to the third putative zinc-binding domain from the transcription factor NZF-1 (hereafter NZF-13) has been synthesized and characterized. The W-visible absorption spectroscopic properties of the cobalt(II) complex of this peptide demonstrate that metal binding is tetrahedral, and the position of the visible absorption bands suggests coordination by three cysteinates and one histidine. To identify which of the two conserved histidine residues acts a metal-binding residue, two histidine to alanine variant peptides were also synthesized. Both variant peptides bound cobalt(II) in a tetrahedral fashion; replacement of the first of the two histidines has a somewhat larger effect on the detailed shape of the absorption spectral features than does replacement of the second histidine. These results suggest that the metal-coordinating residues (italicized) are Cys-X-4-Cys-X-4-His-X-7-His-X-5-Cys. However, simultaneous substitution of both histidine residues with alanine generated a peptide with much more dramatically affected metal binding properties. These observations suggests that the relatively modest effects observed for the singly substituted peptides may be due to metal interactions involving the remaining histidine. Because of these phenomena, further studies will be required to establish more conclusively the roles of the two histidine residues in metal binding and the potential significance of the apparent alternative histidine coordination.