Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin
The coiled coil protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, such as its ability to form filaments in vitro, the proteins length, sequence comparisons and the presence of a coiled coil discontinuity called the "stutter". Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by two-fold symmetry that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine crosslinking we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. Comparison of our crescentin filament structure with current models of IF proteins and their filaments revealed similar coiled coil dimer formation as well as an absence of overall polarity. IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures. SIGNIFICANCE STATEMENTCrescentin is a coiled coil protein that is required for the crescent cell shape of bacteria such as Caulobacter crescentus. Crescentin shares biochemical and cytoskeletal properties with intermediate filament (IF) proteins, which form the third major class of cytoskeletal proteins in eukaryotes. To better understand the relationship between crescentin and IF proteins, and the filaments they form, we have determined the three-dimensional structure of crescentin filaments by cryo-EM. This revealed the full-length structure of the parallel coiled coil dimer of crescentin and how dimers come together laterally and longitudinally, to form a non-polar, octameric filament. Differences in filament architecture highlight the versatility of intermediate filament-like proteins across the tree of life.