Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
J Mol Biol. 2011 Dec 16;414(5):639-53. doi: 10.1016/j.jmb.2011.10.022. Epub 2011 Oct 18.
The positive transcription elongation factor P-TEFb mediates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb is negatively regulated by the cellular protein Hexim1 (hexamethylene bisacetamide-inducible protein 1), which is highly conserved in higher eukaryotes. The C-terminal coiled-coil domain of Hexim1 recognizes the Cyclin T subunit of P-TEFb, whereas a central PYNT motif is required to inhibit the cyclin-dependent kinase Cdk9 by a yet unknown mechanism. Here, the crystal structure of the Cyclin T-binding domain (TBD) of human Hexim1 was determined at 2.1 Å resolution using a deletion mutant of three residues in its central stammer motif. The structure showed a continuous parallel coiled-coil domain of nine hepta-repeats with a preceding helix encompassing up to 15 residues. Two uncommon residues at heptad a positions in the N-terminal part of the coiled-coil structure, Lys284 and Tyr291, stabilize the preceding helix by a tight intermolecular hydrogen bond network with residues of the opposing chain. These interactions delineate a characteristic turn between both helices that is supposed to mediate binding to Cyclin T1. Stabilization of the coiled-coil domain by deletion of the stammer region was confirmed by NMR spectroscopic and backbone dynamic analyses analyzing wild-type TBD and three mutant variants. This study thus provides structural insights into the recognition of the regulator protein Hexim1 by P-TEFb and the modulation of coiled-coil dynamics by specific discontinuities.
正转录延伸因子 P-TEFb 通过磷酸化 RNA 聚合酶 II 的 C 端结构域来介导从转录起始到有效延伸的转变。P-TEFb 受到细胞蛋白 Hexim1（六亚甲基双乙酰胺诱导蛋白 1）的负调控，Hexim1 在高等真核生物中高度保守。Hexim1 的 C 端卷曲螺旋结构域识别 P-TEFb 的 Cyclin T 亚基，而中央 PYNT 基序则通过未知机制抑制细胞周期蛋白依赖性激酶 Cdk9。在这里，使用其中央 stammer 基序中三个残基缺失的突变体，以 2.1Å 的分辨率测定了人 Hexim1 的 Cyclin T 结合结构域（TBD）的晶体结构。结构显示了一个连续的平行卷曲螺旋结构域，由九个七肽重复组成，前面有一个螺旋，最多包含 15 个残基。在卷曲螺旋结构的 N 端部分的七肽 a 位置的两个不常见的残基，Lys284 和 Tyr291，通过与对面链的残基形成紧密的分子间氢键网络来稳定前面的螺旋。这些相互作用在两个螺旋之间划定了一个特征性的转弯，这可能介导与 Cyclin T1 的结合。通过删除 stammer 区域稳定卷曲螺旋结构域，通过 NMR 光谱学和分析野生型 TBD 和三个突变变体的骨架动力学分析得到了证实。因此，这项研究提供了对 P-TEFb 识别调节蛋白 Hexim1 以及特定不连续性对卷曲螺旋动力学调节的结构见解。
