Isogawa Yasushi, Kon Takahide, Inoue Takeshi, Ohkura Reiko, Yamakawa Hisashi, Ohara Osamu, Sutoh Kazuo
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Tokyo 153-8902, Japan.
Biochemistry. 2005 Apr 26;44(16):6190-6. doi: 10.1021/bi0475931.
Myosin XVIII is the recently identified 18th class of myosins, and its members are composed of a unique N-terminal domain, a motor domain with an unusual sequence around the ATPase site, one IQ motif, a segmented coiled-coil region for dimerization, and a C-terminal globular tail. To gain insight into the functions of this unique myosin, we characterized its human homologue, MYO18A, focusing on the functional roles of the characteristic N-terminal domain that contains a PDZ module known to mediate protein-protein interaction. GFP-tagged full-length and C-terminally truncated MYO18A molecules that were expressed in HeLa cells exhibited colocalization with actin filaments. Chemical cross-linking of these molecules showed that they form stable dimers as expected from their putative coiled-coil tails. Cosedimentation of the various types of truncated MYO18A constructs with actin filaments indicated the presence of an ATP-insensitive actin-binding site in the N-terminal domain. Further studies on truncated constructs of the N-terminal domain indicated that this actin-binding site is located outside the PDZ module, but within the middle region of this domain, which does not show any homology with the known actin-binding motifs. These results imply that this dimeric myosin might stably cross-link actin filaments by two ATP-insensitive actin-binding sites at the N-terminal domains for higher-order organization of the actin cytoskeleton.
肌球蛋白XVIII是最近发现的第18类肌球蛋白，其成员由一个独特的N端结构域、一个在ATP酶位点周围具有异常序列的马达结构域、一个IQ模体、一个用于二聚化的分段卷曲螺旋区域以及一个C端球状尾部组成。为了深入了解这种独特肌球蛋白的功能，我们对其人类同源物MYO18A进行了表征，重点关注包含已知介导蛋白质-蛋白质相互作用的PDZ模块的特征性N端结构域的功能作用。在HeLa细胞中表达的绿色荧光蛋白标记的全长和C端截短的MYO18A分子与肌动蛋白丝共定位。这些分子的化学交联表明，正如预期的那样，它们从假定的卷曲螺旋尾部形成稳定的二聚体。各种截短的MYO18A构建体与肌动蛋白丝的共沉降表明在N端结构域中存在一个对ATP不敏感的肌动蛋白结合位点。对N端结构域截短构建体的进一步研究表明，这个肌动蛋白结合位点位于PDZ模块之外，但在该结构域的中间区域内，该区域与已知的肌动蛋白结合基序没有任何同源性。这些结果表明，这种二聚体肌球蛋白可能通过N端结构域中的两个对ATP不敏感的肌动蛋白结合位点稳定地交联肌动蛋白丝，以实现肌动蛋白细胞骨架的高级组织。
