Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.
Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.
复制标题
DOI：
10.1074/jbc.m111.218669
复制
发表时间：
2011-06-17
期刊：
影响因子：
--
通讯作者：
Sellers JR
中科院分区：
文献类型：
--
作者：
Guzik-Lendrum S;Nagy A;Takagi Y;Houdusse A;Sellers JR
编码果蝇肌球蛋白 - 18的基因很复杂，可能产生六种可变剪接的mRNA。这种肌球蛋白的一个主要特征是其N末端的PDZ结构域，它存在于一些预测的可变剪接产物中。为了探究这种蛋白质的生化特性，我们构建了两个类似最小运动结构域（MMD）的构建体，一个包含N末端的PDZ（肌球蛋白 - 18 M - PDZ）结构域，另一个不包含（肌球蛋白 - 18 M - ΔPDZ）。这两个构建体在杆状病毒/Sf9系统中表达。结果表明，果蝇肌球蛋白 - 18与超家族中的大多数其他肌球蛋白有很大差异。这两个MMD构建体都没有肌动蛋白激活的MgATP酶活性，甚至都不结合ATP。肌球蛋白 - 18 M - PDZ和M - ΔPDZ蛋白都与肌动蛋白结合，其Kd值分别为2.61和1.04μm，但即使在高肌动蛋白浓度下，也只有约50 - 75%的蛋白质与肌动蛋白结合。这些肌动蛋白结合实验中未结合的蛋白质再次显示出60%的最大饱和度，这表明在体外果蝇肌球蛋白 - 18存在肌动蛋白结合和非肌动蛋白结合构象之间的平衡。ATP对结合亲和力和亚化学计量结合都没有显著影响。在三珠实验中对单分子的光镊研究显示，肌球蛋白 - 18运动结构域与肌动蛋白丝的相互作用是短暂的。综合来看，这些数据表明这种差异很大的运动结构域可能作为一种肌动蛋白束缚蛋白发挥作用。
The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with Kd values of 2.61 and 1.04 μm, respectively, but only about 50–75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein.