肌动蛋白,在细胞结构和功能中起着至关重要的作用,与各种结合蛋白相互作用,特别是肌球蛋白。在哺乳动物中,肌动蛋白由六个同工型组成,总体上表现出高水平的序列保守性和结构相似性。因此,在与肌球蛋白结合的结构研究中,肌动蛋白亚型的选择被认为不重要。然而,最近的高分辨率结构研究发现了肌动蛋白同工型的N端细微的结构差异,表明每个肌动蛋白同工型都可能与肌球蛋白同工型进行特定的相互作用。在这项研究中,我们旨在探索这种可能性,特别是通过了解不同肌动蛋白同工型对与肌球蛋白7A相互作用的影响。首先,我们比较了使用相同类型的肌动蛋白同工型与使用cryo-EM阐明的高分辨率丝状骨骼α-肌动蛋白(3.5µ)结构的报道的肌动蛋白结构。通过这种比较,我们证实,肌球蛋白亚型的多样性导致与肌动蛋白N端相互作用的差异,肌球蛋白肌动蛋白结合位点的环2直接与肌动蛋白N端相互作用。随后,借助多序列比对,我们观察到不同肌球蛋白同工型的第2环长度有显著差异。我们预测,环2中的这些长度差异可能会导致结构变化,从而影响与肌动蛋白N末端的相互作用。对于肌球蛋白7A,发现循环2很短,使用骨骼α-肌动蛋白和蛋白质复合物预测证实了环2与肌动蛋白N末端之间的相互作用。预测表明,环2中存在的带正电荷的残基与肌动蛋白亚结构域1的酸性贴片残基D24和D25静电相互作用,而没有观察到与超过此的肌动蛋白N-末端的相互作用。此外,使用各种肌动蛋白同工型产生的肌动球蛋白-7A预测模型的分析始终产生相同的结果,而与所用肌动蛋白同工型的类型无关。这项研究的结果表明,肌动蛋白同工型N末端的细微结构差异不太可能影响短环2肌球蛋白7A的结合结构。我们的发现有望为未来的肌动蛋白和肌球蛋白的高分辨率结构结合研究提供更深入的了解。
Actin, which plays a crucial role in cellular structure and function, interacts with various binding proteins, notably myosin. In mammals, actin is composed of six isoforms that exhibit high levels of sequence conservation and structural similarity overall. As a result, the selection of actin isoforms was considered unimportant in structural studies of their binding with myosin. However, recent high-resolution structural research discovered subtle structural differences in the N-terminus of actin isoforms, suggesting the possibility that each actin isoform may engage in specific interactions with myosin isoforms. In this study, we aimed to explore this possibility, particularly by understanding the influence of different actin isoforms on the interaction with myosin 7A. First, we compared the reported actomyosin structures utilizing the same type of actin isoforms as the high-resolution filamentous skeletal α-actin (3.5 Å) structure elucidated using cryo-EM. Through this comparison, we confirmed that the diversity of myosin isoforms leads to differences in interaction with the actin N-terminus, and that loop 2 of the myosin actin-binding sites directly interacts with the actin N-terminus. Subsequently, with the aid of multiple sequence alignment, we observed significant variations in the length of loop 2 across different myosin isoforms. We predicted that these length differences in loop 2 would likely result in structural variations that would affect the interaction with the actin N-terminus. For myosin 7A, loop 2 was found to be very short, and protein complex predictions using skeletal α-actin confirmed an interaction between loop 2 and the actin N-terminus. The prediction indicated that the positively charged residues present in loop 2 electrostatically interact with the acidic patch residues D24 and D25 of actin subdomain 1, whereas interaction with the actin N-terminus beyond this was not observed. Additionally, analyses of the actomyosin-7A prediction models generated using various actin isoforms consistently yielded the same results regardless of the type of actin isoform employed. The results of this study suggest that the subtle structural differences in the N-terminus of actin isoforms are unlikely to influence the binding structure with short loop 2 myosin 7A. Our findings are expected to provide a deeper understanding for future high-resolution structural binding studies of actin and myosin.