Mashhoon N, Carmel G, Pflugrath J W, Kuret J
Biophysics Program, Ohio State University Medical School, Columbus 43210, USA.
Arch Biochem Biophys. 2001 Mar 1;387(1):11-9. doi: 10.1006/abbi.2000.2241.
The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4%. Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes. Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A. In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme. Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding. The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft. The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate. The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft. In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.
测定了来自酿酒酵母的cAMP依赖性蛋白激酶催化亚基的截短变体TPK1δ在无配体状态下的结构，分辨率为2.8 Å，并将其精修至晶体学R因子为19.4%。将该结构与其完全结合配体的哺乳动物同源物的结构进行比较，发现了一个由两个球状叶组成的高度保守的蛋白质折叠结构。在每个叶内，Cα位置的均方根偏差平均约等于0.9 Å。此外，在每种酶的C末端结构域中发现了一个磷酸苏氨酸残基。对这两种结构的进一步比较表明，配体结合伴随着一系列构象变化。第一个变化是一个叶相对于另一个叶发生14.7度的刚体旋转，导致活性位点裂隙关闭。第二个变化仅影响富含甘氨酸的核苷酸结合环，该环移动约3 Å以进一步关闭活性位点并捕获核苷酸底物。第三个变化定位于与配体和配体结合裂隙直接接触的C末端片段。除了解析无配体酶的构象外，该模型还表明，cAMP依赖性蛋白激酶的显著特征在漫长的进化过程中是保守的。
