PDF(Pubmed)
Abstract：
Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act as nucleotide sensors, can substantially determine the kinetic signature of a myosin, i.e., to either perform fast movement or enable long-range transport and tension generation. Switch-2 essentially contributes to the ATP hydrolysis reaction and determines product release. With few exceptions, class-1 myosin harbor a tyrosine in the switch-2 consensus sequence DIYGFE, at a position where class-2 myosins and a selection of myosins from other classes have a substitution. Here, we addressed the role of the tyrosine in switch-2 of class-1 myosins as potential determinant of the duty ratio. We generated constitutively active motor domain constructs of two class-1 myosins from the social amoeba Dictyostelium discoideum, namely, Myo1E, a high duty ratio myosin and Myo1B, a low duty ratio myosin. In Myo1E we introduced mutation Y388F and in Myo1B mutation F387Y. The detailed functional characterization by steady-state and transient kinetic experiments, combined with in vitro motility and landing assays revealed an almost reciprocal relationship of a number of critical kinetic parameters and equilibrium constants between wild-type and mutants that dictate the lifetime of the strongly actin-attached states of myosin. The Y-to-F mutation increased the duty ratio of Moy1B by almost one order of magnitude, while the introduction of the phenylalanine in switch-2 of Myo1E transformed the myosin into a low duty ratio motor. These data together with structural considerations propose a role of switch-2 in fine-tuning ADP release through a mechanism, where the class-specific tyrosine together with surrounding residues contributes to the coordination of Mg2+ and ADP. Our results highlight the importance of conserved switch-2 residues in class-1 myosins for efficient chemo-mechanical coupling, revealing that switch-2 is important to adjust the duty ratio of the amoeboid class-1 myosins for performing movement, transport or gating functions.
摘要：
虽然肌球蛋白共享一个结构保守的运动结构域，活性位点元件的单个氨基酸变异，包括P环，开关-1和开关-2，作为核苷酸传感器，可以基本上确定肌球蛋白的动力学特征，即，执行快速运动或使远程运输和张力的产生。Switch-2基本上有助于ATP水解反应并决定产物释放。除了少数例外,1类肌球蛋白在开关2共有序列DIYGFE中含有酪氨酸，在2类肌球蛋白和来自其他类的肌球蛋白具有替代的位置。这里,我们讨论了酪氨酸在1类肌球蛋白开关-2中作为占空比的潜在决定因素的作用.我们从盘基网柄变形虫中产生了两种1类肌球蛋白的组成性活性运动域构建体，即,Myo1E,高占空比肌球蛋白和Myo1B，低占空比肌球蛋白。在Myo1E中，我们引入了突变Y388F，在Myo1B中引入了突变F387Y。通过稳态和瞬态动力学实验进行详细的功能表征，结合体外运动和着陆测定，揭示了野生型和突变体之间的许多关键动力学参数和平衡常数几乎是相互关系，这决定了肌球蛋白强烈肌动蛋白附着状态的寿命。Y到F突变使Moy1B的占空比增加了几乎一个数量级，而在Myo1E的开关-2中引入苯丙氨酸将肌球蛋白转化为低占空比电机。这些数据与结构考虑一起提出了switch-2在通过一种机制微调ADP释放中的作用，其中类别特异性酪氨酸与周围残基一起有助于Mg2和ADP的配位。我们的结果强调了1类肌球蛋白中保守的switch-2残基对于有效的化学-机械偶联的重要性，揭示了switch-2对于调整变形虫1类肌球蛋白的占空比以进行运动很重要，运输或门控功能。
