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首页> 外文期刊>Biochemistry >Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting
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Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting

机译：通过蛋白质足迹绘制底物诱导的cAMP依赖性蛋白激酶的构象变化

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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. [References: 50]
机译：结合底物后，cAMP依赖性蛋白激酶（cAPK）的催化亚基（C）发生明显的诱导构象变化，从而导致催化作用。对于游离的脱辅酶而言，平衡有利于更开放和可延展的构象，而C，MgATP和20个残基的抑制剂肽[PKI（5-24）]的三元复合物则具有紧密和封闭的构象[Zheng，J.，et al。 。 （1993）Protein Sci。 2，1559]。尚不清楚单独配体中的任一个是否负责此构象转换或是否需要两者。另外，催化亚基协同结合MgATP和抑制剂肽。目前还没有定义这种协同作用的结构基础。使用Fe-EDTA介导的蛋白质足迹技术，通过绘制C的溶剂可及表面和结构动力学图，探究了与MgATP和热稳定蛋白激酶抑制剂（PKI）结合相关的构象变化。酶明显不同于三元复合物。此外，单独结合MgATP会引起局部和全局的广泛构象变化，包括富含甘氨酸的环，连接小叶和大叶的接头，催化环，Mg2 +定位环，激活环和F螺旋。这些变化类似于在三元复合物中所见的变化，与从开放构象到更封闭构象的转变相一致，并且可能反映了与催化和产物释放有关的运动。相反，C.PKT的足迹模式类似于游离C，表明构象变化最小。通过将平衡转移到更紧密的构象，MgATP的结合会“引发”该酶，使其准备好对接PKI，并为MgATP和PKI之间的协同作用提供了解释。 [参考：50]

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