Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family.
Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.
Yang
Sung-Tae
ST
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Jeon
Jae-Hyuck
JH
Kim
Yangmee
Y
Shin
Song Yub
SY
Hahm
Kyung-Soo
KS
Kim
Jae Il
JI
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D054804:Cathelicidins
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D008051:Lipid Bilayers; D010743:Phospholipids; C086301:porcine myeloid antibacterial peptide 23; D054804:Cathelicidins