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The alpha-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (BBCL)ORCID iD: 0000-0003-1241-8888
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
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2012 (English)In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 43, no 4, 1471-1483 p.Article in journal (Refereed) Published
Abstract [en]

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 alpha-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg(7) or Glu(15) 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(15) substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic alpha-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 alpha-defensin salt-bridge facilitates adoption of the characteristic alpha-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.

Place, publisher, year, edition, pages
2012. Vol. 43, no 4, 1471-1483 p.
Keyword [en]
Defensin, Cryptdin-4, Crp4, Salt-bridge, Structure, Folding, Proteolytic stability
National Category
Organic Chemistry
Research subject
Chemistry, Biochemistry; Chemistry, Organic Chemistry
Identifiers
URN: urn:nbn:se:lnu:diva-22288DOI: 10.1007/s00726-012-1220-3ISI: 000309070700007OAI: oai:DiVA.org:lnu-22288DiVA: diva2:565152
Available from: 2012-11-06 Created: 2012-11-06 Last updated: 2017-04-18Bibliographically approved

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Andersson, Håkan S.Haugaard-Kedström, Linda M.Bengtsson, ElinaRosengren, K. Johan
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