title: A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP.
abstract: In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.
authors.affiliation: Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Vanden Broeck
authors.affiliation: Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
authors.forename: Edwige
authors.initials: E
authors.lastname: Van der Heiden
authors.affiliation: Centre for Protein Engineering, Institut de Physique B5a, University of Liège, Sart Tilman, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Sauvage
authors.affiliation: Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Dauvin
authors.affiliation: Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Joris
authors.affiliation: Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
authors.forename: Colette
authors.initials: C
authors.lastname: Duez
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism; D004926:Escherichia coli; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D004150:Dipeptidases; C019989:DD-carboxypeptidase-endopeptidase; D008239:Lysine