Design of a protein kinase-inducible domain
Design of a protein kinase-inducible domain
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DOI：
10.1021/ja057692h
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发表时间：
2006-05-03
影响因子：
15
通讯作者：
Zondlo, NJ
中科院分区：
文献类型：
--
作者：
Balakrishnan, S;Zondlo, NJ
蛋白质磷酸化是一种重要的调控策略。新的工具是必要的，可以用来询问和响应蛋白激酶和磷酸酶的活动。我们已经使用蛋白质设计来开发蛋白质基序，称为蛋白激酶诱导结构域，其结构取决于其磷酸化状态。基于EF手钙结合环，关键设计元素是用丝氨酸残基替换结构关键的Glu残基，其以双齿方式结合金属，丝氨酸残基预期在磷酸化时紧密结合金属，但在未磷酸化时结合不良。该设计包括EF手共有序列，在残基7处的色氨酸以敏化镧系元素发光，以及丝氨酸/苏氨酸激酶的识别序列。设计的肽，其中包含蛋白激酶PKA，PKC，或MAP激酶Erk的最小底物识别基序，形成复合物与Tb 3+磷酸化时，显示出强的Tb 3+发光发射在544 nm，但显示弱发光时，不磷酸化。磷酸化的荧光变化与所述激酶传感器中观察到的荧光变化相当或更大。位点特异性镧系元素结合通过NMR与抗磁性和顺磁性金属确认。激酶诱导结构域肽包含可表达的序列，潜在地使它们能够用作蛋白激酶活性的遗传编码标签。该基序是通用的，并且可能适用于大多数丝氨酸/苏氨酸激酶。
Protein phosphorylation is a critical regulatory strategy. New tools are necessary which may be used to interrogate and are responsive to the activities of protein kinases and phosphatases. We have used protein design to develop a protein motif, termed a protein kinase-inducible domain, whose structure is dependent on its phosphorylation state. Based on an EF hand calcium-binding loop, the key design element is the replacement of a structurally critical Glu residue, which binds metal in a bidentate manner, with a serine residue, which is expected to bind metal tightly when phosphorylated but poorly when not phosphorylated. The design comprises an EF hand consensus sequence, a tryptophan at residue 7 to sensitize lanthanide luminescence, and the recognition sequence of a serine/threonine kinase. Designed peptides, which contain minimal substrate recognition motifs of the protein kinases PKA, PKC, or the MAP kinase Erk, form complexes with Tb3+when phosphorylated, showing strong Tb3+luminescence emission at 544 nm, but show weak luminescence when not phosphorylated. The change in fluorescence on phosphorylation is comparable to or greater than that observed in described kinase sensors. Site-specific lanthanide binding was confirmed by NMR with diamagnetic and paramagnetic metals. The kinase-inducible domain peptides comprise an expressible sequence, potentially enabling their use as genetically encoded tags of protein kinase activity. The motif is general and potentially applicable to the majority of serine/threonine kinases.