title: The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity.
abstract: The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.
authors: Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Spain.
authors: Rubén
authors: R
authors: Cebrián
authors: Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Spain.
authors: Manuel
authors: M
authors: Martínez-Bueno
authors: Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Spain.
authors: Eva
authors: E
authors: Valdivia
authors: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors: Armando
authors: A
authors: Albert
authors: Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Spain.
authors: Mercedes
authors: M
authors: Maqueda
authors: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid, Spain. Electronic address: xmjose@iqfr.csic.es.
authors: María José
authors: MJ
authors: Sánchez-Barrena
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001430:Bacteriocins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D019281:Dimerization; D057927:Hydrophobic and Hydrophilic Interactions; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D055672:Static Electricity; D014461:Ultracentrifugation
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D009841:Oligonucleotides; C440289:bacteriocin AS-48, Enterococcus faecalis
keywords: Antimicrobial peptide; Bacteriocin; Lipid binding protein; Membrane bilayer; X-ray crystallography