Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting
Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting
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DOI：
10.1021/bi981057p
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发表时间：
1998-10-06
期刊：
影响因子：
2.9
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Cheng, XD;Shaltiel, S;Taylor, SS
cAMP依赖性蛋白激酶（cAPK）的催化亚基（C）在与底物结合时经历显著的诱导构象变化，从而导致催化。对于游离脱辅基酶，平衡有利于更开放和可塑的构象，而C、MgATP和20-残基抑制剂肽的三元复合物[PKI（5-24）]采用紧密和封闭的构象[Zheng，J.，等人（1993）Protein Sci. 2，1559]。目前尚不清楚，单独的任何配体的结合是负责这种构象转换或是否两者都是必需的。此外，催化亚基协同结合MgATP和抑制剂肽。这种协同作用的结构基础目前也没有定义。利用Fe-EDTA介导的蛋白质足迹技术，通过绘制C.游离酶的构象与三元复合物有明显区别。此外，结合MgATP单独诱导广泛的构象变化，包括本地和全球的甘氨酸丰富的循环，连接器连接的小和大的叶片，催化环，Mg 2+定位环，激活环，和F螺旋。这些变化，类似于在三元复合物中看到的那些，与从开放到更封闭的构象的转变一致，并且可能反映了与催化和产物释放相关的运动。相比之下，C.PKT的足迹模式类似于游离C，表明最小的构象变化。MgATP的结合，通过将平衡转移到一个更封闭的构象，“引发”酶，使其准备对接的PKI和MgATP和PKI之间的协同作用提供了一个解释。
Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKT resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.