Highly potent p21WAF1-derived peptide inhibitors of CDK-mediated pRb phosphorylation:: Delineation and structural insight into their interactions with cyclin A
Highly potent p21WAF1-derived peptide inhibitors of CDK-mediated pRb phosphorylation:: Delineation and structural insight into their interactions with cyclin A
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DOI：
10.1034/j.1399-3011.2002.21014.x
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发表时间：
2002-11-01
期刊：
影响因子：
--
通讯作者：
Lane, DP
中科院分区：
文献类型：
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作者：
Zheleva, DI;McInnes, C;Lane, DP
肿瘤抑制蛋白p21（WAF 1）在调节真核细胞周期进程中起着重要作用。通过与G1期和S期CDK复合物的结合，它调节视网膜母细胞瘤蛋白（pRb）和E2 F转录因子的活化。p21对CDK/细胞周期蛋白复合物的识别至少部分是通过与细胞周期蛋白亚基上的结合沟的蛋白质-蛋白质相互作用而发生的。相同的凹槽已被证明参与大分子CDK底物的募集，包括pRb和E2 F。因此，阻断该募集位点可以防止CDK底物的识别和随后的磷酸化，并提供了一种恢复p21样肿瘤抑制的治疗方法。从p21的C-末端细胞周期蛋白结合结构域开始，我们已经确定了最小和最佳的生物活性（152）HAKRRLIF（159）肽序列对CDK蛋白激酶的抑制作用，其中pRb是底物。组蛋白H1的磷酸化，但是，它不包含一个可识别的细胞周期蛋白结合基序，不受影响。详细的结构-活性关系研究表明，该序列中的决定簇是残基Arg（155），Leu（157）和Phe（159），并更完整地定义了细胞周期蛋白结合基序的组成。在短合成肽抑制剂的情况下，用Ala残基替换天然Ser（153）后，获得了153效力的显著增加，并且显著地，该突变导致与全长重组p21（其具有CDK 2和细胞周期蛋白A结合位点）相当的与CDK 2/细胞周期蛋白A的亲和力。比较了来自已知与细胞周期蛋白相互作用的各种蛋白质的肽的效力和选择性。构建了细胞周期蛋白沟和HAKRRLIF肽之间复合物的分子模型。该模型解释了所观察到的肽结构-活性关系，包括占据疏水口袋的LIF序列的效力增强。此外，它提供了一般的见解，分子间的相互作用，管理细胞周期蛋白沟识别，并奠定了基础的肽模拟抑制剂的CDKs的发展。
The tumour suppressor protein p21(WAF1) plays a central role in regulating eukaryotic cell-cycle progression. Through its association with G1- and S-phase CDK complexes it regulates activation of the retinoblastoma protein (pRb) and E2F transcription factors. Recognition of CDK/cyclin complexes by p21 occurs, at least in part, through a protein-protein interaction with a binding groove on the cyclin subunit. The same groove has been shown to be involved in the recruitment of macromolecular CDK substrates, including pRb and E2F. Blocking of this recruitment site therefore prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of p21-like tumour suppression. Starting from the C-terminal cyclin-binding domain of p21 we have identified the minimal and optimized bioactive (152)HAKRRLIF(159) peptide sequence with respect to CDK protein kinase inhibition where pRb is the substrate. The phosphorylation of histone H1, however, which does not contain a recognizable cyclin-binding motif, was unaffected. Detailed structure-activity relationship investigations revealed that the determinants within this sequence are residues Arg(155), Leu(157) and Phe(159) and more completely define the composition of the cyclin-binding motif. A marked increase in 153 potency was obtained upon replacement of the native Ser(153) with an Ala residue in the context of short synthetic peptide inhibitors and significantly, this mutation resulted in comparable affinity with CDK2/cyclin A as does the full-length recombinant p21 (which has CDK2 and cyclin A binding sites). Peptides derived from various proteins known to interact with cyclins were compared for potency and selectivity. A molecular model of the complex between the cyclin groove and the HAKRRLIF peptide was constructed. This model accounts for the observed peptide structure-activity relationships, including the potency enhancement of the LIF sequence occupying the hydrophobic pocket. Furthermore, it provides generic insights into molecular interactions governing cyclin groove recognition and lays the foundation for the development of peptidomimetic inhibitors of CDKs.