Interaction of the transmembrane domain of lysis protein E from bacteriophage ?X174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

摘要

The molecular target for the bacteriolytic E protein from bacteriophage ?X174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37?aa peptide Epep, containing the N-terminal transmembrane α-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of Epep was found by circular dichroism (CD) spectroscopy to be 100?% α-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, Epep was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC50 0.15?μM), implying a strong interaction between E and SlyD. Epep was found to inhibit E. coli MraY activity when assayed in membranes (IC50 0.8?μM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein–protein interaction is formed between E and MraY at a site within the transmembrane region.