Brown N R, Noble M E, Lawrie A M, Morris M C, Tunnah P, Divita G, Johnson L N, Endicott J A
Laboratory of Molecular Biophysics, Department of Biochemistry, and Oxford Centre for Molecular Sciences, University of Oxford, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Biol Chem. 1999 Mar 26;274(13):8746-56. doi: 10.1074/jbc.274.13.8746.
We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.
我们利用酿酒酵母的细胞周期蛋白依赖性激酶激活激酶1（CAK1）制备了用于结晶的磷酸化细胞周期蛋白依赖性蛋白激酶2（CDK2），并采用微量接种技术培养出了晶体。CAK1对单体人CDK2的磷酸化作用比对二元CDK2 - 细胞周期蛋白A复合物的磷酸化作用更有效。磷酸化的CDK2表现出组蛋白H1激酶活性，约为完全激活的磷酸化CDK2 - 细胞周期蛋白A复合物所观察到活性的0.3%。荧光测量表明，苏氨酸160磷酸化增加了CDK2对组蛋白底物和ATP的亲和力，并降低了其对ADP的亲和力。相比之下，CDK2的磷酸化对其与细胞周期蛋白A的亲和力影响可忽略不计。已在2.1埃分辨率下解析了磷酸化CDK2和未磷酸化CDK2的ATP结合形式的晶体结构。这些结构相似，主要差异出现在激活片段，该片段在磷酸化的CDK2中是无序的。磷酸化CDK2中激活片段的更大流动性以及缺乏自发结晶表明，磷酸化的CDK2可能采取几种不同的移动状态。这些状态中的大多数可能对应于无活性构象，但一小部分磷酸化的CDK2可能处于活性构象，从而解释了所观察到的基础活性。
