Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain
Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain
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DOI：
10.1039/c9ob00502a
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发表时间：
2019-04-28
影响因子：
3.2
通讯作者：
Zondlo, Neal J.
中科院分区：
文献类型：
--
作者：
Gao, Feng;Thornley, Blair S.;Zondlo, Neal J.
蛋白激酶和磷酸酶调节蛋白质的结构和功能，进而调节细胞的活动。对蛋白质磷酸化反应的新型蛋白质和蛋白质模体的开发为探索单个蛋白激酶的功能及其激活和下调对细胞内的影响提供了新的途径。在这里，我们开发了一个对蛋白质磷酸化反应的最小基序，称为最小蛋白激酶诱导结构域。可编码的蛋白质基序包括一个7或8个残基序列(DKDADXW或DKDADXXW)，来自EF-手性钙结合结构域，这是结合Tb所必需的，但不是充分的，与蛋白质磷酸化位点(第9位的Ser或Thr)结合，当磷酸化时，完成金属结合基序。因此，该基序与金属结合较差，当未磷酸化时显示出微弱的Tb发光。磷酸化后，该多肽与金属的亲和力显著提高，并显示出强劲的Tb发光。磷酸化导致Tb的发光增加高达23倍。开发了小至9个残基的最小磷酸化依赖基序(DKDADGWIS)。对这个La(III)中心点状磷肽络合物的核磁共振谱证实，尽管EF-手中没有通常存在的保守的Glu12，但结合的方式与EF-手中类似。通过将分子设计与已知的蛋白激酶识别序列相结合，开发出了对蛋白激酶A(PKA：DKDADRRW(S/PS)IIAK)、蛋白激酶C(PKC：DKDADGWI(T/PT)FRRKA)和酪蛋白激酶1(CK1：DKDADDWA(S/PS)I)的磷酸化反应做出响应的最小蛋白激酶诱导结构域。在HeLa细胞提取物中PKA的磷酸化被定量，在544 nm处观察到4.4倍的荧光(Tb发光)。优化的最小基序包括1、3和5位的交替天冬氨酸残基，以及7位的主链羰基结合；2位的赖氨酸提供静电平衡，在没有磷酸化的情况下减少结合；4位的丙氨酸促进在EF手的该位置观察到的铝构象；7或8位的色氨酸用于敏化Tb的发光；9位的磷酸化位点与丝氨酸或苏氨酸。6；7或8位的残基；以及10位或更晚的残基可以改变以提供激酶的特异性。在CK1反应肽中，原Tb结合基序中的酸性残基被用作激酶识别序列的一部分。因此，这项工作为设计紧凑的磷酸化响应Tb结合基序提供了基本规则，并有可能进一步应用于响应其他蛋白质翻译后修饰的基序。
Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23x increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(III) center dot phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4x increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the aL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.