Clapperton Julie A, Martin Stephen R, Smerdon Stephen J, Gamblin Steven J, Bayley Peter M
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Biochemistry. 2002 Dec 17;41(50):14669-79. doi: 10.1021/bi026660t.
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 relieve autoinhibition. 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 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable 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 binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the 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 residues associated with the inhibitory pseudosubstrate sequence.
钙饱和钙调蛋白（CaM）通过与钙调蛋白依赖性蛋白激酶I（CaMKI）C端调节序列中的一个区域结合，解除自身抑制，从而直接激活CaMKI。通过X射线晶体学以1.7埃分辨率确定了CaM与CaMKI的25个残基肽段（残基294 - 318）形成的高亲和力复合物的结构。复合物形成后，CaMKI肽段呈α螺旋构象，而CaM结构域连接区的变化使其N端和C端结构域都能围绕肽段螺旋缠绕。靶肽残基Trp - 303（与CaM C端结构域相互作用）和Met - 316（与CaM N端结构域相互作用）确定了1 - 14的结合模式。此外，肽段上的两个碱性区域与CaM形成互补电荷相互作用。CaM - 肽段的亲和力约为1 pM，而CaM - 激酶复合物的亲和力为30 nM，这表明CaM激活自身抑制的CaMKI需要对CaM结合序列与酶的其余部分之间的相互作用进行高能量的破坏。我们提供了生化和结构证据，表明CaM的两个结构域都参与了激活过程：虽然C端结构域对调节序列表现出紧密结合，但N端结构域对于激活是必需的。我们的晶体结构还使我们能够确定完整的CaM结合序列。靶肽段的残基Lys - 296和Phe - 298直接与CaM相互作用，表明自身抑制序列和CaM结合序列之间存在重叠。因此，激酶激活机制涉及CaM与与抑制性假底物序列相关的残基结合。
