Resolving the structure and assembly of the honeybee silk heterotetrameric coiled coil.

Abstract

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.