Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA 95064, USA.
J Mol Biol. 2011 Aug 19;411(3):520-8. doi: 10.1016/j.jmb.2011.05.045. Epub 2011 Jun 17.
Cks (cyclin-dependent kinase subunit) proteins are essential eukaryotic cell cycle regulatory proteins that physically associate with cyclin-dependent kinases (Cdks) to modulate their activity. Cks proteins have also been studied for their ability to form domain-swapped dimers by exchanging β-strands. Domain swapping is mediated by a conserved β-hinge region containing two proline residues. Previous structural studies indicate that Cks in its dimer form is unable to bind Cdk, suggesting that the monomer-dimer equilibrium of Cks may have an effect on Cks-mediated Cdk regulation. We present the crystal structure of a proline-to-alanine mutant Saccharomyces cerevisiae Cks protein (Cks1 P93A) that preferentially adopts the monomer conformation but surprisingly fails to bind Cdk. Comparison of the Cks1 P93A structure to that of other Cks proteins reveals that Pro93 is critical for stabilizing a multiple β-turn structure in the hinge region that properly positions an essential Cdk-binding residue. Additionally, we find that these β-turn formations, conserved in Cks homologs, have implications for the mechanism and preferentiality of strand exchange. Together, our observations suggest that the conservation of Cks hinge-region prolines reflects their functions in forming a Cdk binding interface and that the ability of these prolines to control partitioning between monomer and dimer is a consequence of the β-turn networks within the hinge.
细胞周期蛋白依赖性激酶亚基（Cks）蛋白是真核细胞周期调控所必需的蛋白，其通过与细胞周期蛋白依赖性激酶（Cdks）结合来调节它们的活性。Cks 蛋白也因其通过交换β-链形成结构域交换二聚体的能力而被研究。结构域交换由含有两个脯氨酸残基的保守β-铰链区域介导。先前的结构研究表明，Cks 以二聚体形式无法结合 Cdk，这表明 Cks 的单体-二聚体平衡可能对 Cks 介导的 Cdk 调节有影响。我们展示了一种脯氨酸到丙氨酸突变的酿酒酵母 Cks 蛋白（Cks1 P93A）的晶体结构，该突变体优先采用单体构象，但令人惊讶的是，它无法结合 Cdk。将 Cks1 P93A 结构与其他 Cks 蛋白的结构进行比较，发现 Pro93 对于稳定铰链区域中的多个β-转角结构至关重要，该结构正确定位了一个必需的 Cdk 结合残基。此外，我们发现这些β-转角结构在 Cks 同源物中保守，这对交换的机制和偏好性有影响。综上所述，我们的观察结果表明，Cks 铰链区域脯氨酸的保守性反映了它们在形成 Cdk 结合界面中的功能，而这些脯氨酸控制单体和二聚体之间的分配的能力是铰链内β-转角网络的结果。
