Improving a natural CaMKII inhibitor by random and rational design.
Improving a natural CaMKII inhibitor by random and rational design.
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DOI：
10.1371/journal.pone.0025245
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Bayer KU
中科院分区：
文献类型：
--
作者：
Coultrap SJ;Bayer KU
CaM-KIIN已发展为有效、选择性和有效地抑制Ca 2 +/钙调蛋白（CaM）依赖性蛋白激酶II（CaMKII）的刺激和自主活性（IC 50 ≤ 100 nM）。CN类肽，来自抑制区的CaM-KIIN，提供了强大的新工具，研究CaMKII功能。这项研究的目的是确定CaMKII抑制所需的残基，并评估人工突变是否可以进一步提高进化过程中达到的效力。首先，通过在生物化学测定中确定截短肽对CaMKII活性的影响来鉴定具有完全抑制效力的最小区域（CN 19）。然后，突变CN 19的单个残基。大多数单独的Ala取代降低了CaMKII抑制的效力，然而，P3 A、K13 A和R14 A增加了效力。重要的是，该初始Ala扫描表明R11周围区域与CaMKII底物结合位点的特异性相互作用，其被用于进一步的合理诱变以产生优化的假底物序列。事实上，优化的肽CN 19 〇的效力提高了> 250倍（IC 50 <0.4 nM），并且CN 19 〇具有紧密结合抑制剂的特征。CaMKII相对于CaMKI的选择性类似地提高（对于CN 19 〇几乎提高至100，000倍）。磷酸模拟S12 D突变降低效力，表明通过细胞信号传导调节的潜力。与该残基在抑制中的重要性一致，大多数其他S12突变也显著降低效价，然而，突变为V或Q则不会。这些结果为研究CaMKII功能提供了改进的研究工具，并表明进化微调CaM-KIIN不是为了CaMKII抑制的最大效力，而是为了可能对信号转导的动态调节最佳的较低效力。
CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution. First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not. These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction.
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影响因子：
34.7
作者：
Lee, Yong-Seok;Silva, Alcino J.
通讯作者：
Silva, Alcino J.
影响因子：
56.9
作者：
De Koninck, P;Schulman, H
通讯作者：
Schulman, H
DOI：
10.1073/pnas.0805408105
发表时间：
2008-12-30
影响因子：
11.1
作者：
Alev, Cantas;Urschel, Stephanie;Dermietzel, Rolf
通讯作者：
Dermietzel, Rolf
DOI：
10.1016/s0169-328x(99)00131-x
发表时间：
1999-06-18
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
Bayer, KU;Löhler, J;Harbers, K
通讯作者：
Harbers, K
影响因子：
16.2
作者：
Barria, A;Malinow, R
通讯作者：
Malinow, R