, Volume 71, Issue 1, pp 61–69 | Cite as

Did cis- and trans-defensins derive from a common ancestor?

  • Weiping Zhou
  • Bin Gao
  • Shunyi ZhuEmail author
Original Article


Defensins are small, cysteine-rich, cationic antimicrobial peptides, serving as effectors of the innate immune system and modulators of the adaptive immune system. They extensively exist in multicellular organisms and are divided into cis and trans according to their disulfide bridge connectivity patterns. It has been proposed that these two types of defensins convergently originated from different ancestors. Here, we report the discovery of a structural signature involved in the formation of the cysteine-stabilized α-helix/β-sheet (CSαβ) fold of the cis-defensins in some trans-β-defensins, with only one amino acid indel (CXC vs. CC. C, cysteine; X, any amino acid). The indel of the X residue in the structural signature provides a possible explanation as to why cis- and trans-defensins possess different folds and connectivity patterns of disulfide bridges formed in evolution. Although our attempt to convert the structure type of a present-day trans-defensin with the X residue deleted was unsuccessful due to the low solubility of the synthetic peptide, a combination of data from structural signature, function, and phylogenetic distribution suggests that these defensins may have descended from a common ancestor. In this evolutionary scenario, we propose that a progenitor cis-scaffold might gradually evolve into a trans-defensin after deleting the X residue in specific lineages. This proposal adds a new dimension to more deeply studying the evolutionary relationship of defensins with different folds and of other distantly related proteins.


Antimicrobial peptide Disulfide bridge Structural signature Fold change Evolution 


Author’s contributions

S.Z. conceived and designed the research. W.Z. performed sequence and structural analyses. G.B. performed oxidative refolding experiments of peptides. W.Z., B.G., and S.Z. jointly wrote the paper.


This work was supported by the National Natural Science Foundation of China (Grant Nos. 31870766 and 31570773) to S.Z.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Bontems F, Roumestand C, Gilquin B, Menez A, Toma F (1991) Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. Science 254:1521–1523CrossRefGoogle Scholar
  2. Boulanger N, Bulet P, Lowenberger C (2006) Antimicrobial peptides in the interactions between insects and flagellate parasites. Trends Parasitol 22:262–268CrossRefGoogle Scholar
  3. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250. CrossRefGoogle Scholar
  4. Carvalho Ade O, Gomes VM (2009) Plant defensins--prospects for the biological functions and biotechnological properties. Peptides 30:1007–1020. CrossRefGoogle Scholar
  5. Cordes MH, Walsh NP, McKnight CJ, Sauer RT (1999) Evolution of a protein fold in vitro. Science 284:325–328CrossRefGoogle Scholar
  6. De Leeuw E et al (2010) Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II. FEBS Lett 584:1543–1548. CrossRefGoogle Scholar
  7. Dias Rde O, Franco OL (2015) Cysteine-stabilized alphabeta defensins: from a common fold to antibacterial activity. Peptides 72:64–72. CrossRefGoogle Scholar
  8. Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF, Lehrer RI (1985) Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 76:1427–1435. CrossRefGoogle Scholar
  9. Grishin NV (2001) Fold change in evolution of protein structures. J Struct Biol 134:167–185. CrossRefGoogle Scholar
  10. He Y, Chen Y, Alexander PA, Bryan PN, Orban J (2012) Mutational tipping points for switching protein folds and functions. Structure 20:283–291. CrossRefGoogle Scholar
  11. Hoover DM, Wu Z, Tucker K, Lu W, Lubkowski J (2003) Antimicrobial characterization of human beta-defensin 3 derivatives. Antimicrob Agents Chemother 47:2804–2809CrossRefGoogle Scholar
  12. Hughes AL (1999) Evolutionary diversification of the mammalian defensins. Cell Mol Life Sci 56:94–103CrossRefGoogle Scholar
  13. Ingles-Prieto A, Ibarra-Molero B, Delgado-Delgado A, Perez-Jimenez R, Fernandez JM, Gaucher EA, Sanchez-Ruiz JM, Gavira JA (2013) Conservation of protein structure over four billion years. Structure 21:1690–1697. CrossRefGoogle Scholar
  14. Krishnakumari V, Sharadadevi A, Singh S, Nagaraj R (2003) Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and biophysical properties. Biochemistry 42:9307–9315. CrossRefGoogle Scholar
  15. Lehrer RI, Ganz T (2002) Defensins of vertebrate animals. Curr Opin Immunol 14:96–102CrossRefGoogle Scholar
  16. Li D, Zhang L, Yin H, Xu H, Trask JS, Smith DG, Li Y, Yang M, Zhu Q (2014) Evolution of primate alpha and theta defensins revealed by analysis of genomes. Mol Biol Rep 41:3859–3866. CrossRefGoogle Scholar
  17. Liu L, Zhao C, Heng HH, Ganz T (1997) The human beta-defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry. Genomics 43:316–320. CrossRefGoogle Scholar
  18. Mattar EH, Almehdar HA, Yacoub HA, Uversky VN, Redwan EM (2016) Antimicrobial potentials and structural disorder of human and animal defensins. Cytokine Growth Factor Rev 28:95–111. CrossRefGoogle Scholar
  19. Meier S, Jensen PR, David CN, Chapman J, Holstein TW, Grzesiek S, Ozbek S (2007) Continuous molecular evolution of protein-domain structures by single amino acid changes. Curr Biol 17:173–178. CrossRefGoogle Scholar
  20. Oeemig JS, Lynggaard C, Knudsen DH, Hansen FT, Nørgaard KD, Schneider T, Vad BS, Sandvang DH, Nielsen LA, Neve S, Kristensen HH, Sahl HG, Otzen DE, Wimmer R (2012) Eurocin, a new fungal defensin: structure, lipid binding, and its mode of action. J Biol Chem 287:42361–42372. CrossRefGoogle Scholar
  21. Saito T, Kawabata SI, Shigenaga T, Takayenoki Y, Cho J, Nakajima H, Hirata M, Iwanaga S (1995) A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity. J Biochem 117:1131–1137CrossRefGoogle Scholar
  22. Sass V, Schneider T, Wilmes M, Korner C, Tossi A, Novikova N, Shamova O, Sahl HG (2010) Human beta-defensin 3 inhibits cell wall biosynthesis in staphylococci. Infect Immun 78:2793–2800. CrossRefGoogle Scholar
  23. Schmitt P, Wilmes M, Pugnière M, Aumelas A, Bachère E, Sahl HG, Schneider T, Destoumieux-Garzón D (2010) Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II. J Biol Chem 285:29208–29216. CrossRefGoogle Scholar
  24. Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, Pag U, Jansen A, Nielsen AK, Mygind PH, Raventos DS, Neve S, Ravn B, Bonvin AMJJ, de Maria L, Andersen AS, Gammelgaard LK, Sahl HG, Kristensen HH (2010) Plectasin, a fungal defensin, targets the bacterial cell wall precursor lipid II. Science 328:1168–1172. CrossRefGoogle Scholar
  25. Schroeder BO, Wu Z, Nuding S, Groscurth S, Marcinowski M, Beisner J, Buchner J, Schaller M, Stange EF, Wehkamp J (2011) Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1. Nature 469:419–423. CrossRefGoogle Scholar
  26. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005) The FoldX web server: an online force field. Nucleic Acids Res 33:W382–W388. CrossRefGoogle Scholar
  27. Semple F, Dorin JR (2012) Beta-defensins: multifunctional modulators of infection, inflammation and more? J Innate Immun 4:337–348. CrossRefGoogle Scholar
  28. Semple CA, Gautier P, Taylor K, Dorin JR (2006) The changing of the guard: molecular diversity and rapid evolution of beta-defensins. Mol Divers 10:575–584. CrossRefGoogle Scholar
  29. Shafee TM, Lay FT, Hulett MD, Anderson MA (2016) The defensins consist of two independent, convergent protein Superfamilies. Mol Biol Evol 33:2345–2356. CrossRefGoogle Scholar
  30. Shafee TM, Lay FT, Phan TK, Anderson MA, Hulett MD (2017) Convergent evolution of defensin sequence, structure and function. Cell Mol Life Sci 74:663–682. CrossRefGoogle Scholar
  31. Silva PM, Goncalves S, Santos NC (2014) Defensins: antifungal lessons from eukaryotes. Front Microbiol 5:97. Google Scholar
  32. Stewart KL, Nelson MR, Eaton KV, Anderson WJ, Cordes MH (2013) A role for indels in the evolution of Cro protein folds. Proteins 81:1988–1996. CrossRefGoogle Scholar
  33. Suarez-Carmona M, Hubert P, Delvenne P, Herfs M (2015) Defensins: “simple” antimicrobial peptides or broad-spectrum molecules? Cytokine Growth Factor Rev 26:361–370. CrossRefGoogle Scholar
  34. Sun YM, Liu W, Zhu RH, Goudet C, Tytgat J, Wang DC (2002) Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis. J Pept Res 60:247–256CrossRefGoogle Scholar
  35. Tamaoki H, Miura R, Kusunoki M, Kyogoku Y, Kobayashi Y, Moroder L (1998) Folding motifs induced and stabilized by distinct cystine frameworks. Protein Eng 11:649–659CrossRefGoogle Scholar
  36. Tang KY, Wang X, Wan QH, Fang SG (2018) A crucial role of paralogous beta-defensin genes in the Chinese alligator innate immune system revealed by the first determination of a Crocodilia defensin cluster. Dev Comp Immunol 81:193–203. CrossRefGoogle Scholar
  37. Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, Ouellette AJ, Selsted ME (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286:498–502CrossRefGoogle Scholar
  38. Tassanakajon A, Somboonwiwat K, Amparyup P (2015) Sequence diversity and evolution of antimicrobial peptides in invertebrates. Dev Comp Immunol 48:324–341. CrossRefGoogle Scholar
  39. Teng L, Gao B, Zhang S (2012) The first chordate big defensin: identification, expression and bioactivity. Fish Shellfish Immunol 32:572–577. CrossRefGoogle Scholar
  40. Torres AM, Kuchel PW (2004) The beta-defensin-fold family of polypeptides. Toxicon 44:581–588. CrossRefGoogle Scholar
  41. Tysoe C, Williams LK, Keyzers R, Nguyen NT, Tarling C, Wicki J, Goddard-Borger ED, Aguda AH, Perry S, Foster LJ, Andersen RJ, Brayer GD, Withers SG (2016) Potent human alpha-amylase inhibition by the beta-defensin-like protein helianthamide. ACS Cent Sci 2:154–161. CrossRefGoogle Scholar
  42. Wang G, Li X, Wang Z (2016) APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Res 44:D1087–D1093. CrossRefGoogle Scholar
  43. Wu Y, Gao B, Zhu S (2017) New fungal defensin-like peptides provide evidence for fold change of proteins in evolution. Biosci Rep 37:BSR20160438. CrossRefGoogle Scholar
  44. Wu Z, Hoover DM, Yang D, Boulegue C, Santamaria F, Oppenheim JJ, Lubkowski J, Lu W (2003) Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3. Proc Natl Acad Sci U S A 100:8880–8885. CrossRefGoogle Scholar
  45. Yamaguchi Y, Peigneur S, Liu J, Uemura S, Nose T, Nirthanan S, Gopalakrishnakone P, Tytgat J, Sato K (2016) Role of individual disulfide bridges in the conformation and activity of spinoxin (alpha-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom. Toxicon 122:31–38. CrossRefGoogle Scholar
  46. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schröder JM, Wang JM, Howard OM, Oppenheim JJ (1999) Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525–528CrossRefGoogle Scholar
  47. Yang M, Zhang C, Zhang X, Zhang MZ, Rottinghaus GE, Zhang S (2016) Structure-function analysis of avian beta-defensin-6 and beta-defensin-12: role of charge and disulfide bridges. BMC Microbiol 16:210. CrossRefGoogle Scholar
  48. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395. CrossRefGoogle Scholar
  49. Zhu S (2007) Evidence for myxobacterial origin of eukaryotic defensins. Immunogenetics 59:949–954. CrossRefGoogle Scholar
  50. Zhu S (2008) Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSalphabeta defensins. Mol Immunol 45:828–838. CrossRefGoogle Scholar
  51. Zhu S, Gao B (2013) Evolutionary origin of beta-defensins. Dev Comp Immunol 39:79–84. CrossRefGoogle Scholar
  52. Zhu S, Gao B, Tytgat J (2005) Phylogenetic distribution, functional epitopes and evolution of the CSalphabeta superfamily. Cell Mol Life Sci 62:2257–2269. CrossRefGoogle Scholar
  53. Zou J, Mercier C, Koussounadis A, Secombes C (2007) Discovery of multiple beta-defensin like homologues in teleost fish. Mol Immunol 44:638–647. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Group of Peptide Biology and Evolution, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of ZoologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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