Vanden Broeck Arnaud, Van der Heiden Edwige, Sauvage Eric, Dauvin Marjorie, Joris Bernard, Duez Colette
Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.
Centre for Protein Engineering, Institut de Physique B5a, University of Liège, Sart Tilman, Belgium.
PLoS One. 2015 Oct 13;10(10):e0140082. doi: 10.1371/journal.pone.0140082. eCollection 2015.
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.
在枯草芽孢杆菌C1类青霉素结合蛋白（PBP）PBP4a中，四个成簇的赖氨酸（K）残基，即K86、K114、K119和K265，从结构域II突出。通过定点诱变将这些氨基酸替换为谷氨酰胺（Q）残基，得到了Mut4KQ PBP4a。当在没有其预测的Sec信号肽的情况下在大肠杆菌中产生时，发现野生型（WT）PBP4a主要与宿主细胞质膜相关，而Mut4KQ PBP4a在很大程度上仍未结合。纯化后，比较了这两种蛋白质与枯草芽孢杆菌膜结合的能力。结果与在大肠杆菌中获得的结果相似：在体外，发现与枯草芽孢杆菌膜相互作用的WT PBP4a的百分比远高于Mut4KQ PBP4a。对枯草芽孢杆菌膜提取物中PBP4a的免疫检测表明，这种PBP的一种加工形式（由其大小表明）与枯草芽孢杆菌细胞质膜相关。在PBP4a中不存在任何两亲性肽的情况下，结构域II表面的正电荷冠可能负责该PBP的细胞定位及其与细胞质膜的附着。
