Interaction of the transmembrane domain of lysis protein E from bacteriophage phiX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD.
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 E(pep), containing the N-terminal transmembrane alpha-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of E(pep) was found by circular dichroism (CD) spectroscopy to be 100 % alpha-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, E(pep) was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC(50) 0.15 microM), implying a strong interaction between E and SlyD. E(pep) was found to inhibit E. coli MraY activity when assayed in membranes (IC(50) 0.8 microM); 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.
Mendel
Sharon
S
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Holbourn
Joanne M
JM
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Schouten
James A
JA
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Bugg
Timothy D H
TDH
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001407:Bacillus / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001433:Bacteriolysis; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008954:Models, Biological; D010455:Peptides / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups); D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C029555:E protein, bacteriophage X174; D029968:Escherichia coli Proteins; D010455:Peptides; C085606:SlyD protein, E coli; D014764:Viral Proteins; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria; D019696:Peptidylprolyl Isomerase