Specificity of the transport of Lipid II by FtsW in Escherichia coli [Membrane Biology]

April 7th, 2014 by Mohammadi, T., Sijbrandi, R., Lutters, M., Verheul, J., Martin, N., den Blaauwen, T., de Kruijff, B., Breukink, E.

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to get insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II; the important building block of the bacterial cell wall; and its inner membrane localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (R145 and K153) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants as manifested by their inability to complement a temperature sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analogue by about 274 Da, resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted pore-like structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.