Department of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.
Biomolecular Research Group, School of Natural and Applied Sciences, Canterbury Christ Church University, Canterbury, United Kingdom.
J Biol Chem. 2020 Oct 16;295(42):14522-14535. doi: 10.1074/jbc.RA120.014684. Epub 2020 Aug 19.
We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate ( ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity ( ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 ( ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.
我们研究了肌球蛋白催化结构域内四个替代外显子编码区域之一的生化和生物物理特性。该区域由替代外显子 3a 和 3b 编码，并包含 N 端β桶的一部分。通过在天然慢胚胎体壁（EMB）和快间接飞行肌肌球蛋白同工型（IFI）之间交换编码外显子 3 的区域，生成了嵌合肌球蛋白构建体（IFI-3a 和 EMB-3b）。我们发现这种交换改变了肌球蛋白 S1 头的动力学特性。在没有肌动蛋白的情况下，每个嵌合体的 ADP 释放速率（ ）与天然同工型完全相反。稳态数据也表明存在相互转移，IFI-3a 的基础和肌动蛋白激活的 ATP 酶活性与野生型（WT）IFI 相比降低，而对于 EMB-3b，这些值与野生型（WT）EMB 相比增加。在肌动蛋白存在的情况下，IFI-3a 的 ADP 亲和力（ ）与 IFI 相比没有变化，但 EMB-3b 的 ADP 亲和力增加，并且与 IFI 值接近。两种外显子 3 嵌合体的肌球蛋白 S1 与肌动蛋白的 ATP 诱导解离（ ）降低。同源建模，结合最近报道的 EMB 晶体结构，表明肌球蛋白头部的外显子 3 编码区域是核苷酸结合口袋（嘌呤结合环）和必需轻链之间通讯途径的一部分，强调了这个可变的 N 端结构域在调节肌球蛋白同功型肌球蛋白横桥动力学方面的重要作用，特别是在肌球蛋白同工型的力感测特性方面。
