Ulke-Lemée Annegret, Ishida Hiroaki, Chappellaz Mona, Vogel Hans J, MacDonald Justin A
Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada.
Biochim Biophys Acta. 2014 Sep;1844(9):1580-90. doi: 10.1016/j.bbapap.2014.05.011. Epub 2014 Jun 4.
The smoothelin-like 1 protein (SMTNL1) is a modulator of smooth and skeletal muscle contractility and can bind to calmodulin and tropomyosin. Calmodulin is the major calcium sensor of eukaryotic cells and it can cycle between calcium-free (apo-CaM) and calcium-bound (Ca-CaM) forms. Bioinformatic screening of the SMTNL1 sequence predicted a second CaM-binding region (CBD1) that is located N-terminal to the previously defined apo-CaM-binding site (CBD2). Pull-down assays, surface plasmon resonance, isothermal calorimetry and NMR techniques were used to determine that CBD1 associated preferentially to Ca-CaM while CBD2 bound preferentially to apo-CaM. Mutation of hydrophobic residues abolished Ca-CaM-binding to CBD1 while acidic residues in CBD2 were necessary for apo-CaM-binding to CBD2. The dissociation constant (Kd) for Ca-CaM-binding to a CBD1 peptide was 26∗10(-6)M while the value for binding to a longer protein construct was 0.5∗10(-6)M. The binding of SMTNL1 to both apo-CaM and Ca-CaM suggests that endogenous CaM is continuously associated with SMTNL1 to allow for quick response to changes in intracellular calcium levels. We also found that the intrinsically disordered N-terminus of SMTNL1 can reduce binding to apo-CaM and increase binding to Ca-CaM. This finding suggests that an additional CaM-binding region may exist and/or that intramolecular interactions between the N-terminus and the folded C-terminus reduce apo-CaM-binding to CBD2. Intriguingly, CBD1 is located close to the SMTNL1 phosphorylation site and tropomyosin-binding region. We discuss the possibility that all three signals are integrated at the region surrounding CBD1.
类平滑肌动蛋白1（SMTNL1）蛋白是平滑肌和骨骼肌收缩性的调节因子，可与钙调蛋白及原肌球蛋白结合。钙调蛋白是真核细胞的主要钙传感器，它能在无钙（脱钙钙调蛋白，apo-CaM）和结合钙（结合钙钙调蛋白，Ca-CaM）两种形式之间循环。对SMTNL1序列进行生物信息学筛选预测出了第二个钙调蛋白结合区域（CBD1），该区域位于先前定义的脱钙钙调蛋白结合位点（CBD2）的N端。通过下拉实验、表面等离子体共振、等温滴定量热法和核磁共振技术确定，CBD1优先与结合钙钙调蛋白结合，而CBD2优先与脱钙钙调蛋白结合。疏水性残基的突变消除了结合钙钙调蛋白与CBD1的结合，而CBD2中的酸性残基是脱钙钙调蛋白与CBD2结合所必需的。结合钙钙调蛋白与CBD1肽的解离常数（Kd）为26×10⁻⁶M，而与更长蛋白质构建体结合的值为0.5×10⁻⁶M。SMTNL1与脱钙钙调蛋白和结合钙钙调蛋白的结合表明，内源性钙调蛋白持续与SMTNL1结合，以便对细胞内钙水平的变化做出快速反应。我们还发现，SMTNL1的内在无序N端可减少与脱钙钙调蛋白的结合并增加与结合钙钙调蛋白的结合。这一发现表明可能存在另一个钙调蛋白结合区域和/或N端与折叠的C端之间的分子内相互作用减少了脱钙钙调蛋白与CBD2的结合。有趣的是，CBD1靠近SMTNL1磷酸化位点和原肌球蛋白结合区域。我们讨论了这三种信号在CBD1周围区域整合的可能性。
