Functional consequences of preorganized helical structure in the intrinsically disordered cell-cycle inhibitor p27Kip1

被引:127
作者
Bienkiewicz, EA
Adkins, JN
Lumb, KJ [1 ]
机构
[1] Colorado State Univ, Dept Biochem & Mol Biol, Ft Collins, CO 80523 USA
[2] Colorado State Univ, Dept Chem, Ft Collins, CO 80523 USA
关键词
D O I
10.1021/bi015763t
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
p27(Kip1) contributes to cell-cycle regulation by inhibiting cyclin-dependent kinase (Cdk) activity. The p27 Cdk-inhibition domain has an ordered conformation comprising an alpha-helix, a 3(10) helix, and beta-structure when bound to cyclin A-Cdk2. In contrast, the unbound p27 Cdk-inhibition domain is intrinsically disordered (natively unfolded) as shown by circular dichroism spectroscopy, lack of chemical-shift dispersion, and negative heteronuclear nuclear Overhauser effects. The intrinsic disorder is not due to the excision of the Cdk-inhibition domain from p27, since circular dichroism spectra of the full-length protein are also indicative of a largely unfolded protein. Both the inhibition domain and full-length p27 are active as cyclin A-Cdk2 inhibitors. Using circular dichroism and proline mutagenesis, we demonstrate that the unbound p27 Cdk-inhibition domain is not completely unfolded. The domain contains marginally stable helical structure that presages the alpha-helix, but not the 31() helix, adopted upon binding cyclin A-Cdk2. Increasing or reducing the stability of the partially preformed alpha-helix in the isolated p27 domain with alanine or proline substitutions did not affect formation of the p27-inhibited cyclin A-Cdk2 complex in energetic terms. However, stabilization of the helix with alanine hindered kinetically the formation of the inhibited complex, suggesting that p27 derives a kinetic advantage from intrinsic structural disorder.
引用
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页码:752 / 759
页数:8
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