Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming

Abstract

AbstractMreB is a bacterial protein belonging to the actin superfamily. It polymerizes into an antiparallel double-stranded filament that generally functions for cell shape determinations by maintaining the cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are responsible for its swimming motility. SpeMreB5 is likely responsible for generating the driving force for the swimming motility. However, molecular profiles involved in the swimming motility are poorly understood. Additionally, SpeMreB3 has distinct sequence features from the other SpeMreBs. Here, we have revealed the structures and polymerization dynamics of SpeMreB3 and SpeMreB5. Both SpeMreBs formed antiparallel double-stranded filaments with different characters; SpeMreB3 formed short filaments with slow polymerization, and SpeMreB5 filaments further assembled into bundle structures such as raft and paracrystal. SpeMreB5 filaments hydrolyzed ATP at a constant rate and were depolymerized immediately after ATP depletion. The Pi release rate of SpeMreB3 was much slower than that of SpeMreB5. Our crystal structure of SpeMreB3 and Pi release measurements of SpeMreB3 and SpeMreB5 mutant variants explain that the cause of the slow Pi release is the lack of the amino acid motif “E … T - X - [DE]”, found in almost all MreBs, which probably takes roles to adjust the position and eliminate a proton of the putative nucleophilic water for γ-Pi of AMPPNP. These results show that SpeMreB3 has unique polymerization dynamics without bundle formations, whereas SpeMreB5 shows bundle formations, and its polymerization dynamics occur in the same manner as other actin superfamily members.Significance StatementMreB, a member of the actin superfamily, is widely conserved as a single copy on the genomes in bacteria having cell walls. It forms filaments and functions as a part of a protein complex for cell wall maintenance. While the number of genes and the cellular function of MreB are widely conserved for all bacterial phyla, several MreBs defying the conventional characteristics have been found. Spiroplasma, wall-less bacteria, have at least five MreBs (MreB1-5) in the genome and are thought to use the MreBs for the swimming motility of the cell. Here, we performed structural and biochemical analyses on two of five MreBs (MreB3 and MreB5), and found unique structural and polymerization features.