Mapping Substrate-Induced Conformational Changes in cAMP-Dependent Protein Kinase by Protein Footprinting
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- 作者：Xiaodong Cheng ; Shmuel Shaltiel ; and Susan S. Taylor
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 6, 1998
- 年：1998
- 卷：37
- 期：40
- 页码：14005 - 14013
- 全文大小：185K
- 年卷期：v.37,no.40(October 6, 1998)
- ISSN：1520-4995
文摘
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 apoenzymeequilibrium 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 thisconformational switch or whether both are required. In addition, the catalytic subunit binds MgATP andinhibitor 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 withthe binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping thesolvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearlydistinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensiveconformational changes, both local and global, that include the glycine-rich loop, the linker connectingthe small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the Fhelix. These changes, similar to those seen in the ternary complex, are consistent with a transition froman open to a more closed conformation and likely reflect the motions that are associated with catalysisand product release. In contrast, the footprinting pattern of C·PKI resembled free C, indicating minimalconformational 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 synergismbetween MgATP and PKI.