The Single Septin from Chlamydomonas reinhardtii Reveals a Polyproline II Helix-based NC-interface as an Ancestral Scaffold for Filament Assembly

To date, the most detailed structural characterization of septins has been undertaken on those from opisthokonts, where heterooligomeric complexes polymerize end-to-end into filaments stabilized by alternating G- and NC-interfaces. These filaments are involved in a wide range of essential intracellular processes involving membranes, cytoskeletal components and other binding partners. Their central GTP-binding G-domain is highly conserved and similar to that seen in small monomeric or dimeric GTP-binding proteins, which normally play roles in cell signaling. However, these small GTPases do not polymerize. How and when during evolution septins gained this unique capability is not fully understood. Here we provide seven new crystal structures of the single septin from the green alga, Chlamydomonas reinhardtii, in the form of different constructs, mutations, complexes and crystal forms. This has allowed us to describe the unusual properties of the NC-interface for the first time. These include a polyproline II helix in place of the conventional α0 helix, an extension to the first three β-strands, a novel polyacidic region not seen in opisthokonts and a flexible α6 helix whose curvature can vary depending on filament formation or not. This unusual NC-interface may represent a relatively unstable, primordial interaction which has subsequently evolved in opisthokonts to incorporate the more stable α0 helix, an event which occurred in parallel with the gene expansion which enabled the formation of their more robust heterofilaments.