Exploring GTP-Driven Conformational Changes in Septins Using High-Resolution Cryo-EM

Abstract

Septins are crucial components of the eukaryotic cytoskeleton, involved in a wide range of cellular processes, including cytokinesis, membrane compartmentalization, and vesicle trafficking. To perform their functions, septins need to form heterofilaments, which subsequently assemble into higher-order structures such as rings, networks, and more. Classified into four groups, septin heterofilaments can be composed of one representative from each group, arranged in a specific order and stabilized by contact interfaces known as the G interface (nucleotide-binding domain (GTP/GDP) and the NC interface (N- and C-terminal domains). In mammals, the heterofilament can be assembled from octamers formed by SEPT2-6-7-9-9-7-6-2, with the NC interface formed by SEPT9-9 being critical for assembling octamers from tetramers. Additionally, this interface has shown conformational changes depending on the bound nucleotide (GTP or GDP), suggesting a communication mechanism between the G interface and the NC interface associated with GTP hydrolysis. Despite their biological significance, this mechanism and its relationship with filament assembly, particularly the role of GTP hydrolysis, remain poorly understood. This project aims to investigate these mechanisms by resolving high-resolution structures of septin complexes using single particle Cryo Electron Microscopy (cryo-EM). By focusing on the central tetramer SEPT7-9-9-7, this research seeks to capture conformational changes in septins during GTP binding and hydrolysis, elucidating the cross-talk between the G and NC interfaces recently proposed by our research group (Mendonça et al. 2024). The recombinant SEPT7-9-9-7 complex will be co-expressed and purified, after which it will be incubated with an excess of non-hydrolyzable GTP analogs (GppNHp or GTP¿S) to induce the exchange of GDP for GTP and thereby obtain structures in both states. These structures will provide unprecedented insights into the dynamic conformational shifts critical to septin filament assembly. Conducting this research in Dr. Bruno Klaholz's laboratory, a leading Cryo-EM laboratory comprising advanced instrumentation at the associated facility, will provide access to state-of-the-art equipment and expertise in structural biology, particularly in the processing of difficult datasets in which the possibility of heterogeneous particles is likely as complete nucleotide exchange may not be achieved (i.e. sorting of GDP and GTP states may be required using advanced image processing). This opportunity will allow me to gain specialized skills in Cryo-EM, directly contributing to Brazil's growing capacity in this field and advancing our group's research efforts, including the use of the recently installed 100 kV Tundra microscope in São Carlos. The findings from this project are expected to have far-reaching implications, offering new models for septin filament dynamics.