Mechanism of sensing antimicrobial peptides by bacteria : towards a structural understanding ; LPS-bound structures of designed peptides : effects of incorporation of β-turn nucleating amino acids (D-Pro and Asn).

Abstract

The Aps system, consisting of a transmembrane sensor protein ApsS and two cytosolic ApsX and ApsR, is responsible for antimicrobial peptides (AMPs) resistance mechanism in Gram-positive bacteria. The ApsS acts as a sensor to have direct interactions with AMPs through its extra-cellular loop connected to two transmembrane (TM) segments. To determine the three-dimensional structures of ApsS and to elucidate the interactions of AMPs with the TM domains of ApsS, the TM regions of Staphylococcus epidermidis ApsS (ApsS-TM) were constructed, expressed and purified for NMR structural studies. Uniformly 15N-labeled of ApsS-TM were overexpressed as a fusion protein with ketosteroid isomerase protein (KSI) in Escherichia coli, and the fusion protein was purified by nickel affinity chromatography under denaturing conditions. Cyanogen bromide (CNBr) cleavage was used to release the ApsS-TM-His6 from the fusion protein. Final recovery of ApsS-TM-His6 was achieved by nickel affinity chromatography under denaturing conditions. High amount and good purity of ApsS-TM-His6 protein were demonstrated. However, the protein was too hydrophobic to be solubilized in acetonitrile and in sodium dodecyl sulfate (SDS) detergents, preventing structural studies by NMR spectroscopy.