Rasson Amy S, Bois Justin S, Pham Duy Stephen L, Yoo Haneul, Quinlan Margot E
Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California Los Angeles, Paul D. Boyer Hall, 611 Charles E. Young Drive East, Box 951570, Los Angeles, CA 90095-1570, USA.
J Mol Biol. 2015 Feb 27;427(4):824-839. doi: 10.1016/j.jmb.2014.09.002. Epub 2014 Sep 16.
The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.
最近发现的一类肌动蛋白成核因子，即WASp同源结构域2（WH2）成核因子，利用单体肌动蛋白结合WH2结构域的串联重复来促进肌动蛋白成核。WH2结构域参与多种肌动蛋白调节活动。从结构上看，它们预计会与肌动蛋白丝内的原聚体间接触发生冲突。因此，它们在成核中的作用的发现令人惊讶。在这里，我们使用果蝇Spire（Spir）作为模型系统，来研究串联的WH2结构域如何使肌动蛋白成核，以及含WH2的成核蛋白与其非成核对应物有何不同。我们发现Spir中的第三个WH2结构域（Spir-C或SC）发挥着独特作用。在一个短的成核构建体（仅包含两个WH2结构域）的情况下，将SC置于N端位置对于最强有力的成核是必需的。我们发现，WH2结构域彼此之间的天然组织对于以正协同性结合肌动蛋白是必要的。我们在SC中鉴定出两个对其活性至关重要的残基。利用这些信息，我们能够将一个弱的合成成核因子转化为一个活性与天然Spir构建体相当的成核因子。最后，我们发现有证据表明，除了单体之外，SC还能结合肌动蛋白丝。
