Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.
Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.
Wu
Zhibin
Z
Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
Hoover
David M
DM
Yang
De
D
Boulègue
Cyril
C
Santamaria
Fanny
F
Oppenheim
Joost J
JJ
Lubkowski
Jacek
J
Lu
Wuyuan
W
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D002630:Chemotactic Factors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D023083:beta-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
D000900:Anti-Bacterial Agents; D002630:Chemotactic Factors; C422950:DEFB103A protein, human; D004220:Disulfides; D023083:beta-Defensins; D003545:Cysteine