Prediction and Dissection of Widely-Varying Association Rate Constants of Actin-Binding Proteins

Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as ka = ka0, where ka0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold ka variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

肌动蛋白（actin）是一种丰度极高的蛋白质，是真核细胞骨架（eukaryotic cytoskeleton）的核心组分之一。其聚合与解聚过程受到多种肌动蛋白结合蛋白（actin-binding proteins）的调控。这类蛋白的功能涵盖肌动蛋白聚合的成核、以1∶1复合物形式螯合G-肌动蛋白（G-actin）等多个维度。这类复合物形成的动力学过程至关重要，其速率常数的变化幅度至少可达三个数量级，这直接决定了其差异化的调控功能。此前，我们开发了用于计算蛋白质结合机制与结合速率常数的瞬态复合物理论（transient-complex theory）。该理论中的瞬态复合物（transient complex）指的是一种结合中间态：此时两个相互结合的蛋白质已处于近天然态的间距与相对取向，但尚未形成天然复合物所特有的短程特异性相互作用。结合速率常数的预测公式为kₐ = kₐ⁰，其中kₐ⁰为通过自由扩散抵达瞬态复合物的基础速率常数，而玻尔兹曼因子（Boltzmann factor）则用于表征长程静电相互作用的偏好性。本研究将瞬态复合物理论应用于七种肌动蛋白结合蛋白与G-肌动蛋白的结合动力学研究。由于分子形状与柔性存在差异，这七种蛋白可分为三类结合机制。它们之间高达1000倍的kₐ差异，主要可归因于静电贡献的显著不同。此外，基础速率常数也存在变化，这源于七种肌动蛋白结合蛋白与G-肌动蛋白所形成结合界面的形状与尺寸各不相同。本研究证实，肌动蛋白结合蛋白可通过多种物理特性途径，调控自身的结合机制与速率常数，以适配不同的调控功能。