Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Gangwon, Republic of Korea.
Research Solution Center, Institute for Basic Science, Daejeon 34126, Republic of Korea.
Molecules. 2024 Jun 23;29(13):2992. doi: 10.3390/molecules29132992.
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.
肌动蛋白在细胞结构和功能中起着至关重要的作用，它与各种结合蛋白相互作用，特别是肌球蛋白。在哺乳动物中，肌动蛋白由六种同工型组成，它们在整体上表现出高度的序列保守性和结构相似性。因此，在肌球蛋白与肌动蛋白结合的结构研究中，肌动蛋白同工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肌动蛋白同工型的 N 端存在细微的结构差异，这表明每种肌动蛋白同工型可能与特定的肌球蛋白同工型发生特异性相互作用。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肌动蛋白同工型的报道的肌球蛋白结构，与使用 cryo-EM 阐明的高分辨率丝状骨骼 α-肌动蛋白（3.5 Å）结构进行比较。通过这种比较，我们证实了肌球蛋白同工型的多样性导致与肌动蛋白 N 端的相互作用不同，并且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，借助多重序列比对，我们观察到不同肌球蛋白同工型中环 2 的长度存在显著差异。我们预测，这些环 2 长度差异可能导致结构变化，从而影响与肌动蛋白 N 端的相互作用。对于肌球蛋白 7A，环 2 非常短，并且使用骨骼 α-肌动蛋白的蛋白质复合物预测证实了环 2 与肌动蛋白 N 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 的酸性斑残基 D24 和 D25 静电相互作用，而与肌动蛋白 N 端的进一步相互作用则未观察到。此外，使用各种肌动蛋白同工型生成的肌球蛋白-7A 预测模型的分析结果无论使用哪种肌动蛋白同工型都产生了相同的结果。这项研究的结果表明，肌动蛋白同工型的 N 端细微结构差异不太可能影响与短环 2 肌球蛋白 7A 的结合结构。我们的研究结果有望为未来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。
