Probing self-association of CetZ1 cytoskeletal protein from Haloferax volcanii

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The cytoskeleton is a dynamic network of proteins, which are required by all cells for cell division, growth, and maintenance of cell shape. A major group of cytoskeleton proteins present in nearly all cells are the tubulin superfamily proteins. Archaea, the third domain of life, encode a great diversity of tubulin superfamily proteins, including FtsZ and the tubulins that are more similar to those in eukaryotes. Recently a new group of cytoskeletal proteins was found in archaea, named “CetZ”, which show characteristics in common with both tubulin and FtsZ and are involved in cell shape regulation. They form dynamic cytoplasmic filaments at or near the cell envelope, which are required for cell shape determination (Duggin et al., 2015). However, the mechanisms by which CetZ proteins lead to remodelling of the cell envelope to modulate cell shape remain unknown. Based on crystal structures of CetZ proteins and their likely manner of self-association, we have initiated a structure-function analysis of CetZ interactions in vivo (De Silva, 2019 PhD Thesis) and, in the present study, in vitro. Point mutations were introduced into the Haloferax volcanii CetZ1 protein, designed to target putative functional interactions in self-association and putative membrane association. In the present study, mutations that disrupt the longitudinal and lateral interactions were selected for the in vitro analysis. Light scattering and TEM were used as an approach to analyse the polymerization cycle and structural features of CetZ polymers, correlating these to the in vivo structures observed by high- and super-resolution fluorescence microscopy. The in vitro studies demonstrated that CetZ1 forms GTP-dependent single-stranded filaments and polymer stability was clearly altered in predicted self-association mutants. The longitudinal interface mutants demonstrated that the GTP binding site and GTPase activity controls the longitudinal interaction of CetZ1 polymer formation by GTP hydrolysis. Lateral interaction mutants showed decreased polymerization ability. The mutation in the M-loop region, revealed that it is crucial for polymer formation. Preliminary investigation into possible CetZ1 lipid membrane binding suggests that CetZ1 can bind to the lipid membrane and modify shape changes in them, dependent on the polymerization ability of CetZ1. These findings have contributed to the understanding of tubulin-like cytoskeleton proteins in archaea, which, by comparison to the cytoskeletons of bacteria and eukaryotes, are expected to provide future insights into cytoskeleton evolution and help reveal fundamental principles of cytoskeletal function across the three domains of life.
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