Department of Chemistry and Biochemistry, University of California (UCLA), Los Angeles, CA, 90095, USA.
California NanoSystems Institute, UCLA, Los Angeles, CA, 90095, USA.
Nat Commun. 2017 Dec 19;8(1):2183. doi: 10.1038/s41467-017-02357-8.
Actin filament assembly and disassembly are vital for cell functions. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin's M44 and M47 residues to induce cellular F-actin disassembly. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin's nucleotide-bound state. Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin. Modeling actin's D-loop region based on our 3.9 Å cryoEM reconstruction suggests that oxidation by Mical reorients the side chain of M44 and induces a new intermolecular interaction of actin residue M47 (M47-O-T351). Site-directed mutagenesis reveals that this interaction promotes Mox-actin instability. Moreover, we find that Mical oxidation of actin allows for cofilin-mediated severing even in the presence of inorganic phosphate. Thus, in conjunction with cofilin, Mical oxidation of actin promotes F-actin disassembly independent of the nucleotide-bound state.
肌动蛋白丝的组装和拆卸对于细胞功能至关重要。MICAL 氧化还原酶是肌动蛋白的重要翻译后效应因子，可立体特异性地氧化肌动蛋白的 M44 和 M47 残基，诱导细胞 F-肌动蛋白解体。在这里，我们表明 Mical 氧化的（Mox）肌动蛋白可以进行极快的（84 个亚基/s）解体，这取决于 F-肌动蛋白的核苷酸结合状态。使用近原子分辨率的 cryoEM 重建和单丝 TIRF 显微镜，我们确定了 Mox-肌动蛋白的两种动态和结构状态。基于我们 3.9Å cryoEM 重建的肌动蛋白 D-环区域的建模表明，Mical 的氧化作用使 M44 的侧链重新定向，并诱导肌动蛋白残基 M47（M47-O-T351）的新分子间相互作用。定点突变显示，这种相互作用促进了 Mox-肌动蛋白的不稳定性。此外，我们发现肌动蛋白的 Mical 氧化作用允许肌球蛋白介导的切割，即使存在无机磷酸盐也是如此。因此，与肌球蛋白结合，Mical 氧化的肌动蛋白促进 F-肌动蛋白解体，而不依赖于核苷酸结合状态。
