Heissler Sarah M, Chinthalapudi Krishna, Sellers James R
From the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8015 and
the Cell Adhesion Laboratory, Department of Integrative Structural and Computational Biology, Scripps Research Institute, Jupiter, Florida 33458.
J Biol Chem. 2017 Nov 3;292(44):18372-18385. doi: 10.1074/jbc.M117.801456. Epub 2017 Sep 7.
Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5-dependent transport processes in cells.
肌球蛋白-5B是一种普遍存在的分子马达，它在内吞循环途径中运输内膜系统的货物囊泡。肌球蛋白-5B功能异常会导致先天性肠病微绒毛包涵体病，突显了其在细胞内稳态中的重要性。在此，我们描述了肌球蛋白-5B与F-肌动蛋白、核苷酸以及吡唑并嘧啶化合物myoVin-1的相互作用。我们发现，单头肌球蛋白-5B是一种中等占空比的马达，其动力学ATP酶循环受磷酸释放的限速。第二个头部的存在会在肌球蛋白-5B二聚体中产生张力和门控作用，通过降低肌动蛋白激活的ADP释放速率使其成为限速步骤，从而改变动力学特征。这种向高占空比马达的动力学转变是肌球蛋白-5B在细胞循环途径中所提出的运输功能的先决条件。此外，我们表明小分子化合物myoVin-1会抑制肌球蛋白-5B的酶活性和功能活性。对肌动蛋白激活的稳态ATP酶活性和滑动速度的部分抑制表明，在探究myoVin-1对细胞中肌球蛋白-5依赖性运输过程的影响时应谨慎使用。
