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Amino Acids

, Volume 43, Issue 4, pp 1471–1483 | Cite as

The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance

  • Håkan S. Andersson
  • Sharel M. Figueredo
  • Linda M. Haugaard-Kedström
  • Elina Bengtsson
  • Norelle L. Daly
  • Xiaoqing Qu
  • David J. Craik
  • André J. OuelletteEmail author
  • K. Johan RosengrenEmail author
Original Article

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-defensin cryptdin-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 Arg7 and Glu15 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 to degradation by proteinases.

Keywords

Defensin Cryptdin-4 Crp4 Salt-bridge Structure Folding Proteolytic stability 

Notes

Acknowledgments

This work was supported in part by the Linnaeus University (KJR and HSA), Australian Research Council and National Medical and Health Research Council (NHMRC) (DJC), and NIH Grants DK044632 and AI059346 (AJO). KJR is an NHMRC Biomedical CDA Fellow. NLD is a Queensland Smart State Fellow. DJC is an NHMRC Professorial Research Fellow. The authors gratefully acknowledge the use of the Queensland NMR Network 900 MHz spectrometer.

Supplementary material

726_2012_1220_MOESM1_ESM.pdf (74 kb)
Supplementary material 1 (PDF 74 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Håkan S. Andersson
    • 3
  • Sharel M. Figueredo
    • 4
    • 5
  • Linda M. Haugaard-Kedström
    • 3
  • Elina Bengtsson
    • 3
  • Norelle L. Daly
    • 2
  • Xiaoqing Qu
    • 4
  • David J. Craik
    • 2
  • André J. Ouellette
    • 4
    Email author
  • K. Johan Rosengren
    • 1
    • 3
    Email author
  1. 1.School of Biomedical SciencesThe University of QueenslandBrisbaneAustralia
  2. 2.Institute for Molecular Bioscience, The University of QueenslandBrisbaneAustralia
  3. 3.School of Natural SciencesLinnaeus UniversityKalmarSweden
  4. 4.Department of Pathology and Laboratory MedicineKeck School of Medicine of USC, USC/Norris Cancer CenterLos AngelesUSA
  5. 5.LifeTechnologies Inc.CarlsbadUSA

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