Membrane binding properties of the cytoskeletal protein bactofilin

Bactofilins are a widespread family of cytoskeletal proteins with important roles in bacterial morpho-genesis, chromosome organization and motility. They polymerize in a nucleotide-independent manner, forming non-polar filaments that are typically associated with the cytoplasmic membrane. Membrane binding was suggested to be mediated by a short N-terminal peptide, but the underlying mechanism and the conservation of this interaction determinant among bacteria remain unclear. Here, we use the bacto-filin homolog BacA of the stalked bacterium Caulobacter crescentus as a model to analyze the membrane-binding behavior of bactofilins. Based on site-directed mutagenesis of the N-terminal region, we identify the full membrane-targeting sequence of BacA (MFSKQAKS) and pinpoint amino acid residues that are critical for its function in vivo and in vitro . Molecular dynamics simulations then provide detailed insight into the molecular mechanism underlying the membrane affinity of this peptide. Collectively, these analyses reveal a delicate interplay between the water exclusion of hydrophobic N-terminal residues, the arrangement of the peptide within the membrane and the electrostatic attraction between positively charged groups in the peptide and negative charges in the phospholipid molecules. A comprehensive bio-informatic analysis shows that the composition and properties of the membrane-targeting sequence of BacA are conserved in numerous bactofilin homologs from diverse bacterial phyla. Importantly, our findings reveal cooperative effects between the membrane-binding and polymerization activities of BacA. Moreover, they demonstrate that both of these activities critically contribute to the recruitment of the BacA client protein PbpC, a membrane-bound cell wall synthase that uses a conserved peptide in its N-terminal cytoplasmic tail to interact with BacA assemblies. Finally, we show that PbpC can functionally replace the endogenous membrane-targeting sequence of BacA when provided at elevated levels in trans , indicating that client proteins can make a significant contribution to the membrane association of bacto-filin polymers. Together, these results unravel the mechanistic underpinnings of membrane binding by bactofilin homologs, thereby illuminating a previously obscure but important aspect in the biology of this cytoskeletal protein family.