Nucleotide dependence in polymerization and membrane remodelling by the bacterial actin mreb5 from spiroplasma citri

Abstract

MreB, the bacterial ancestor of eukaryotic actin, determines the shape of most of the rod-shaped bacteria. Until now, functional studies for MreBs have been carried out for cell-walled rod-shaped bacteria. MreB filaments are said to assist rod shape by their circumferential movement, which is dependent on the peptidoglycan synthesis machinery. However, the role of filament dynamics of MreB polymerization for conferring rod shape is unknown. Spiroplasma is a helical cell-wall-less bacterium, which maintains its cell shape in the absence of cell wall synthesis machinery. Recently, we have shown that MreB5, one of the five MreB paralogs of the cell-wall less bacterium Spiroplasma citri (ScMreB5) is essential for helical shape and motility of the organism. My Ph.D. work aims to characterize ScMreB5 using structural, biochemical, and in vitro reconstitution approach, with a goal to understand the fundamental principles of shape determination by MreB. Structurally, we show that features such as protofilament organization and nucleotide binding pocket are well conserved in ScMreB5. We also demonstrate that filament dynamics of the ATPase deficient mutant ScMreB5E134A is compromised. Thus, the catalytic Glu134 plays a dual role, firstly, by sensing ATP bound state of MreB for filament assembly and secondly, assisting ATP hydrolysis leading to filament disassembly. Interestingly, membrane binding of ScMreB5 is mediated via charge-based interaction and is also dependent on the nucleotide state of the protein. We report that ScMreB5 under different nucleotide conditions can remodel lipid bilayer and lipid tubes. This remodeling ability is dependent on the conformational cycle as it goes through the steps of ATP hydrolysis and nucleotide exchange. Our results are indicative of an allosteric effect of the nucleotide-binding site on membrane binding. Thus, ScMreB5 functions as a prototype to understand membrane remodelling by MreBs independent of the cell-wall synthesis machinery.