Computational design of calmodulin mutants with up to 900-fold increase in binding specificity.
Computational design of calmodulin mutants with up to 900-fold increase in binding specificity.
复制标题
结合特异性提高高达 900 倍的钙调蛋白突变体的计算设计。
DOI：
10.1016/j.jmb.2008.09.053
复制
发表时间：
2009
影响因子：
5.6
通讯作者：
Shifman,JuliaM
中科院分区：
文献类型：
--
作者：
Yosef,Eliyahu;Politi,Regina;Choi,MeeH;Shifman,JuliaM
钙调蛋白（Calmodulin, CaM）是一种普遍存在的第二信使蛋白，在响应Ca2+浓度变化的过程中调节多种结构和功能多样化的靶标。CaM依赖性蛋白激酶II （CaMKII）和钙调神经磷酸酶（CaN）是CaM的主要靶点，在包括突触调节在内的许多细胞功能中发挥相反的作用。由于CaMKII和CaN竞争可用的Ca2+/CaM，这些酶对CaM的不同亲和力对于实现Ca2+信号的平衡至关重要。我们使用计算蛋白设计方法来修改这两个靶点的CaM结合特异性。我们从CaM与CaMKII的CaM结合域复合物的x射线结构出发，通过在CaM序列中引入突变来优化CaM与CaMKII的相互作用。CaM优化使用蛋白质设计程序ORBIT进行，该程序使用改进的能量函数，强调序列选择过程中的分子间相互作用。利用表面等离子体共振技术，实验构建了几种CaM变体，并测试了它们与CaMKII和CaN肽的结合。与野生型CaM相比，大多数CaM突变体对CaMKII肽的亲和力略有增加，而对CaN肽的亲和力则大幅降低。我们的最佳CaM设计显示出对CaMKII肽的结合特异性增加了约900倍，成为通过计算蛋白设计方法在任何蛋白质-蛋白质界面中实现的最高特异性开关。我们的研究结果表明，蛋白质界面的计算重新设计成为改变蛋白质结合亲和力和特异性的可靠方法。
Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca2+concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca2+/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca2+signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein–protein interface through the computational protein design approach. Our results show that computational redesign of protein–protein interfaces becomes a reliable method for altering protein binding affinity and specificity.