Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with cellular factors. Spermine, an endogenous polyamine, promotes and stabilizes tubulin assemblies. Yet, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced tubulin to assemble in vitro into hierarchical architectures, based on a tubulin conical-spiral (TCS) subunit. Using solution X-ray scattering and cryo-TEM, we showed that with progressive increase of spermine concentration, tubulin-dimers assembled into a tubulin helical-pitch (or a short TCS), TCSs, TCS that stacked into tubes through base-to-top packing, antiparallel bundles of TCS tubes in a quasi-hexagonal symmetry, and eventually twisted hexagonal bundles of inverted tubulin tubules. Time-resolved experiments revealed that tubulin assemblies formed at low spermine concentrations were precursors of the assemblies formed at higher spermine concentrations. The results provide insight into the variety of morphologies that tubulin can form, and contribute to our understanding of the fundamental interactions that control the composition and construction of protein-based biomaterials.