AbstractLow copy-number plasmid pLS32 ofBacillus subtilissubsp.nattocontains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB and the centromeric sequenceparN. Similar to the ParMRC partitioning system fromE. coliplasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids, through interactions with AlfB andparN. Since, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of non-bundling AlfA filaments to 3.4 Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin-fold has been accommodated by unique longitudinal and lateral contacts, whilst still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments we obtained a pseudo-atomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly anti-phasic with 8-fold integer helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, signs of the strong evolutionary pressure for simplicity, placed on plasmids: deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerisation, nucleotide hydrolysis and antiparallel doublet formation are retained to fulfil the system's biological raison d'être.Significance StatementProtein filaments perform a vast array of functions inside almost all living cells. Actin-like proteins in archaea and bacteria have previously been found to form a surprising diversity of filament architectures, reflecting their divergent cellular roles. Actin-like AlfA is unique in that it is much smaller than all other filament forming actin-like proteins. With an atomic structure of the AlfA filament, obtained by high-resolution electron cryo-microscopy, we have revealed—at atomic level of detail—how AlfA filaments form dynamic filaments capable of transporting plasmid DNA in cells and how these filaments arrange into antiparallel bundles required for the segregation mechanism.
Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli
as refined in the monoclinic crystal lattice