title: 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 285:8985-8994, 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 287:418-428, 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.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Ting-Wei
authors.initials: TW
authors.lastname: Chang
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Chiu-Feng
authors.initials: CF
authors.lastname: Wang
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Hsin-Jye
authors.initials: HJ
authors.lastname: Huang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Iren
authors.initials: I
authors.lastname: Wang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors.forename: Shang-Te Danny
authors.initials: ST
authors.lastname: Hsu
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan ydliao@ibms.sinica.edu.tw.
authors.forename: You-Di
authors.identifier: http://orcid.org/0000-0001-5527-4736
authors.initials: YD
authors.lastname: Liao
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D015870:Gene Expression; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C060507:outer membrane lipoprotein I, Bacteria; D000249:Adenosine Monophosphate