Structure of the Complex of Calmodulin with the Target Sequence of Calmodulin-Dependent Protein Kinase I: Studies of the Kinase Activation Mechanism
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- 作者：Julie A. Clapperton ; Stephen R. Martin ; Stephen J. Smerdon ; Steven J. Gamblin ; and Peter M. Bayley
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：December 17, 2002
- 年：2002
- 卷：41
- 期：50
- 页码：14669 - 14679
- 全文大小：567K
- 年卷期：v.41,no.50(December 17, 2002)
- ISSN：1520-4995
文摘
Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I(CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieveautoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI(residues 294-318) has been determined by X-ray crystallography at 1.7 Å resolution. Upon complexformation, the CaMKI peptide adopts an 
-helical conformation, while changes in the CaM domain linkerenable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303(interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of bindingas 1-14. In addition, two basic patches on the peptide form complementary charge interactions withCaM. The CaM-peptide affinity is ~1 pM, compared with 30 nM for the CaM-kinase complex, indicatingthat activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactionsbetween the CaM-binding sequence and the rest of the enzyme. We present biochemical and structuralevidence indicating the involvement of both CaM domains in the activation process: while the C-domainexhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystalstructure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 fromthe target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residuesassociated with the inhibitory pseudosubstrate sequence.
-helical conformation, while changes in the CaM domain linkerenable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303(interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of bindingas 1-14. In addition, two basic patches on the peptide form complementary charge interactions withCaM. The CaM-peptide affinity is ~1 pM, compared with 30 nM for the CaM-kinase complex, indicatingthat activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactionsbetween the CaM-binding sequence and the rest of the enzyme. We present biochemical and structuralevidence indicating the involvement of both CaM domains in the activation process: while the C-domainexhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystalstructure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 fromthe target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residuesassociated with the inhibitory pseudosubstrate sequence.