title: The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance.
abstract: Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensincryptdin-4 (Crp4) and peptide variants with mutations at Arg7 or Glu15 residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(150 substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance todegradation by proteinases.
authors.affiliation: School of Natural Sciences, Linnaeus University, 39182 Kalmar, Sweden.
authors.forename: Håkan S
authors.initials: HS
authors.lastname: Andersson
authors.forename: Sharel M
authors.initials: SM
authors.lastname: Figueredo
authors.forename: Linda M
authors.initials: LM
authors.lastname: Haugaard-Kedström
authors.forename: Elina
authors.initials: E
authors.lastname: Bengtsson
authors.forename: Norelle L
authors.initials: NL
authors.lastname: Daly
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Qu
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.forename: André J
authors.initials: AJ
authors.lastname: Ouellette
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry* / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000254:growth & development; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000254:growth & development; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D019879:Paneth Cells / Q000502:physiology; D055550:Protein Stability; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012492:Salts; D014357:Trypsin / Q000737:chemistry; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D011993:Recombinant Fusion Proteins; D012492:Salts; D023084:alpha-Defensins; C401847:cryptdin 4, mouse; D018698:Glutamic Acid; D001120:Arginine; D014357:Trypsin; D020783:Matrix Metalloproteinase 7