ABSTRACT
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 obtain 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 (Arg(145) and Lys(153)) 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 analog 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 porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Membrane Proteins/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Amino Acid Sequence , Arginine/chemistry , Arginine/genetics , Arginine/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biological Transport , Cell Wall/metabolism , Escherichia coli/genetics , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutation , Protein Structure, Secondary , Proteolipids/metabolism , Sequence Homology, Amino Acid , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Uridine Diphosphate N-Acetylmuramic Acid/metabolismABSTRACT
The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.
