Johnston Caitlin L, Jobichen Chacko, Briggs Lyndall J, Michie Michelle, Liu Jian-Wei, Morton Craig J, Warden Andrew C, Sutherland Tara D
Health & Biosecurity Research Unit, CSIRO, Canberra, Australian Capital Territory, Australia.
Centre for Advanced Microscopy, Australian National University, Canberra, Australian Capital Territory, Australia.
Protein Sci. 2025 Aug;34(8):e70230. doi: 10.1002/pro.70230.
Coiled coil structures, first proposed by Crick in the 1950s, are protein structural motifs found across diverse biological systems. Honeybee silk was among the earliest identified coiled coils, with X-ray diffraction studies in the 1960s revealing its characteristic helical packing. Decades of research have provided insights into silk composition and formation, yet the molecular details of its coiled coil assembly and final structure remained unresolved. In this study, we generated a structural model of the tetrameric coiled coil using AlphaFold and validated it with crosslinking mass spectrometry and medium-resolution cryo-electron microscopy. The model reveals that the four proteins (F1-F4) adopt an antiparallel configuration in a defined clockwise arrangement (F1-F3-F2-F4). Furthermore, we experimentally investigated the formation of this coiled coil complex using biochemical techniques, including blue-native PAGE and circular dichroism spectroscopy. The sum of these experimental results and the structural predictions has allowed for the elucidation of key transitional steps in the assembly pathway, suggesting molecular interactions that may drive tetramer formation. These findings support a stepwise assembly model in which F2 and F4 form a stable core, F3 binds to the complex, and F1 initiates formation of the final, highly ordered structure. These structural insights establish a framework for understanding and directing coiled coil assembly, the fundamental building block of honeybee silk. By resolving this structure and its assembly process, this work lays the foundation for future rational design of functional sequences and materials with tailored properties.
卷曲螺旋结构最早由克里克在20世纪50年代提出，是在各种生物系统中发现的蛋白质结构基序。蜜蜂丝是最早被鉴定出的卷曲螺旋之一，20世纪60年代的X射线衍射研究揭示了其特征性的螺旋堆积。数十年的研究为丝的组成和形成提供了见解，但卷曲螺旋组装及其最终结构的分子细节仍未得到解决。在这项研究中，我们使用AlphaFold生成了四聚体卷曲螺旋的结构模型，并用交联质谱和中分辨率冷冻电子显微镜对其进行了验证。该模型表明，四种蛋白质（F1-F4）以定义的顺时针排列（F1-F3-F2-F4）采用反平行构型。此外，我们使用生化技术，包括蓝色非变性聚丙烯酰胺凝胶电泳和圆二色光谱，对这种卷曲螺旋复合物的形成进行了实验研究。这些实验结果和结构预测的总和使得能够阐明组装途径中的关键过渡步骤，表明可能驱动四聚体形成的分子相互作用。这些发现支持了一种逐步组装模型，其中F2和F4形成一个稳定的核心，F3与该复合物结合，F1启动最终高度有序结构的形成。这些结构见解为理解和指导卷曲螺旋组装建立了一个框架，卷曲螺旋是蜜蜂丝的基本组成部分。通过解析这种结构及其组装过程，这项工作为未来合理设计具有定制特性的功能序列和材料奠定了基础。
