Actin-like filament A (AlfA) and AlfA-driven plasmid segregation

Thesis (Ph.D.)--University of Washington, 2018For a long time, the cytoskeleton was considered an exclusive feature of eukaryotic cells. In 1992, it was proposed that bacteria might contain actin and tubulin homologs. Cell biology and structural biology research within the last two decades led to a paradigm shift in our understanding of the cytoskeleton. Discovery of actin and tubulin homologs in bacteria that can form filaments upon nucleotide binding and their involvement in various cellular processes including cell shape determination, cell division, and DNA segregation led to a reconsideration of the evolutionary history of life. Actin-like filament A (AlfA) is a bacterial actin involved in plasmid segregation in Bacillus subtilis natto, and it is encoded by plasmid pLS32. In this dissertation, I investigate AlfA and the AlfA-driven segregation system, which is composed of the cytomotive AlfA filaments, the DNA-binding protein AlfB and the centromeric DNA sequence parN. We obtained a near-atomic resolution structure of an AlfA filament in the presence of AMP-PNP using cryo-electron microscopy (cryo-EM). We built an atomic model based on the electron density map and the sequence information to reveal the atomic details of filament formation. Sequence analysis suggested that AlfA is missing the canonical actin subdomain IIB, which we confirmed by our atomic model. Lack of subdomain IIB is compensated by increased lateral interaction surface area, which we confirmed by both cryo-EM and hydrogen-deuterium exchange mass spectrometry (HDX-MS). A comparison of AlfA structure with other actins revealed a unique nucleotide binding conformation, which causes AlfA to remain stable after nucleotide hydrolysis. In addition to the filament structure, we reconstituted the segresome (centromeric DNA-adaptor protein) complex in vitro and used it as a tool to investigate AlfA-AlfB interactions. We found that the segresome nucleates AlfA at low stoichiometric ratios while free AlfB increases the effective critical concentration by sequestering AlfA monomers. However, we found that both the segresome and free AlfB can prevent AlfA polymerization at the equimolar ratio and high stoichiometric ratios, respectively. Our crosslinking results suggest that AlfB interacts with AlfA through the same interaction surface to both sequester AlfA monomers and nucleate AlfA filaments