STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF TOC90 AND ITS INTERACTION WITH THE SINGLE SEPTIN CRSEP IN CHLAMYDOMONAS REINHARDTII

Septins are GTP-binding proteins capable of assembling into higher-order structures, such as filaments and rings, playing key roles in membrane organization and cellular architecture. Chlamydomonas reinhardtii possesses a single septin, CrSEP, which is capable of forming filaments. In our previous work, the structural characterization of CrSEP revealed an alternative NC interface, in which a Polyproline II (PPII) helix replaces the α0-helix characteristic of opisthokont septins. This architecture, together with alterations in the electrostatic distribution at the interface, provides evidence of a potential ancestral mechanism of septin polymerization. Despite these advances, the mechanisms that may stabilize or organize CrSEP filaments on the chloroplast surface in C. reinhardtii remain poorly understood. Recently, an interaction was identified between CrSEP and TOC90, a GTPase of the TOC159 family associated with the protein import machinery in the outer chloroplast membrane. This interaction is predicted to involve the GTPase domains of both proteins, and computational structural analyses suggest the participation of the CrSEP G interface. In this work, we aim to structurally and biochemically characterize TOC90 and determine the molecular basis of its interaction with CrSEP. To this end, the GTPase domain of TOC90 (TOC90_G​) was produced in Escherichia coli, purified, and utilized to evaluate its stability, GTPase activity, and oligomeric state using biochemical and biophysical techniques, including thermal shift assays and SEC-MALS. Currently, the CrSEP–TOC90_G​ interaction is being investigated through pull-down assays under various conditions. In parallel, crystallization trials of TOC90_G​ are underway, and co-crystallization of the CrSEP–TOC90_G ​complex will also be attempted. Determining the structure of this complex may reveal the specific residues and interfaces involved, thereby establishing the structural basis of the CrSEP–TOC90 interaction and helping clarify a possible molecular link between CrSEP polymerization and its organization at the chloroplast membrane.