Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides.

ABSTRACT

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 2858985-8994, 2010, http//dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287418-428, 2012, http//dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.