INTERACTIONS OF MONOMERIC RABBIT NEUTROPHIL DEFENSINS WITH BILAYERS -COMPARISON WITH DIMERIC HUMAN DEFENSIN HNP-2

Human antimicrobial neutrophil defensin HNP-2 has been shown to form l arge multimeric pores in pure 1-palmitoyl-2-oleoyl phosphatidylglycero l (POPG) bilayers that lead to all-or-none release of vesicle contents [Wimley et al. (1994) Protein Sci. 3, 1362-1373]. Because human neutr ophil defensins form natural dimers in solution, the question arises a s to the role of dimerization in pore formation. However, the dimers a re so stable that this question is not easily answered directly, Rabbi t neutrophil defensins, whose three-dimensional structures are very si milar to those of human defensins, are monomeric in aqueous solution a nd thus provide an opportunity to test the hypothesis that dimerizatio n may play a role in multimeric pore formation. We therefore examined the interactions of the six known rabbit neutrophil defensins with lar ge unilamellar vesicles (LUV) under the conditions known to lead to st able pore formation by HNP-2. We find that the rabbit defensins bind s trongly to LUVs formed from pure POPG or mixtures of POPG with neutral (zwitterionic) phospholipid but induce leakage of vesicle contents on ly from pure POPG vesicles. Rabbit defensin NP-4 does not cause leakag e under any conditions examined. The remaining defensins, NP-1, NP-2, NP-3A, NP-3B, and NP-5. cause graded release of the contents of purr: POPG vesicles as does a mixture of the six defensins. The graded relea se indicates that the rabbit defensins do not form stable pores in the membrane. This result thus suggests that the structural features of h uman defensins that permit dimer formation in aqueous solution are lik ely to be important in the formation of multimeric ports.