Generation of a megabody toolkit for crystallization and performing cryo-EM of the SOG1 NAC domain

Samenvatting

SOG1 plays a major role in the DNA Damage Response (DDR) of plants, regulating more than hundred genes involved in this process. SOG1 belongs to the NAC (NAM, ATAF1/2 and CUC2) family of transcription factors, which are known for their role in plant development and stress responses. Despite its crucial role in protecting the genomic integrity of plants, no structure of SOG1 and its NAC domain, has been identified yet. Uncovering the structure of the NAC domain, would give us an insight on the mechanism of action of the SOG1 protein. The NAC domain of SOG1 forms a dimer similar to the identified NAC domains. However, the small molecular weight of the NAC dimer (around 40 kDa) possess a challenge for structural studies especially for cryo-EM. Forming crystals for NAC domain of SOG1 is hard due to several issues including protein purity, stability, optimal concentration, sample homogeneity, and identifying suitable crystallization conditions. To address this problem, we generated a megabody toolkit made up of nanobodies against the NAC domain grafted with two scaffold proteins (HopQ and Ygjk) in order to increase the molecular weight and therefore facilitate structural studies for cryo-EM and feasibility of forming crystals. To create these megabodies, 32 nanobodies targeting the SOG1 NAC domain were generated by the VIB nanobody core. In this thesis, 11 nanobodies of 11 different CDR3 groups were cloned into a pHen6c vector and transformed into E. coli WK6 cells for expression. After expressing and purifying all of the selected nanobodies, their ability to bind to the NAC domain was tested using analytical gel filtration and their capacity to bind to the NAC domain in complex with DNA while not interfering with DNA binding was checked with Electron mobility shift assay (EMSA). ITC was also performed which revealed that the molar ratio is 1:1 per Nb and NAC monomer, and that the Kd (dissociation constant) of the Nb to the NAC domain is within the nanomolar range indicating a high affinity. This concluded that only four out of the eleven nanobodies can bind to the NAC domain, and that from these four, only one nanobody does not interfere with the DNA binding of NAC. Then after testing Nb1 and Nb2 with the NTD-NAC, it was revealed that Nb1 is likely blocking the NTD-NAC to bind to DNA. Finally, three of the binding nanobodies were successfully cloned into the megabody frameworks, except Nb6 were it was only cloned in one plasmid. These megabodies can then be further used for crystallization and structure determination through cryo-EM and X-ray crystallography of the SOG1 NAC domain or NTD-NAC domain.

Datum prijs	25 jun. 2024
Originele taal	English