Curvature-Dependent Recognition of Ethanolamine Phospholipids by
Duramycin and Cinnamycin
Kunihiko Iwamoto,* Tomohiro Hayakawa,yMotohide Murate,* Asami Makino,* Kazuki Ito,zTetsuro Fujisawa,z
and Toshihide Kobayashi*y§
*Supra-Biomolecular System Research Group, RIKEN (Institute of Physical and Chemical Research) Frontier Research System, Saitama,
Japan;yLipid Biology Laboratory, RIKEN, Saitama, Japan;zRIKEN SPring-8 Center, Hyogo, Japan; and§INSERM UMR 870, INRA U1235,
INSA-Lyon, University Lyon 1 and Hospices Civils de Lyon, Villeurbanne, France
bind phosphatidylethanolamine (PE). The lipid specificity of duramycin was not established. The present study indicates that
both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids (PE and ethanolamine plasmalogen). Model
membrane study indicates that the binding of duramycin and cinnamycin to PE-containing liposomes is dependent on membrane
curvature, i.e., the lantibiotics bind small vesicles more efficiently than large liposomes. The binding of the lantibiotics to
multilamellar liposomes induces tubulation of membranes, as revealed by electron microscopy and small-angle x-ray scattering.
These results suggest that both duramycin and cinnamycin promote their binding to the PE-containing membrane by deforming
Duramycin is a 19-amino-acid tetracyclic lantibiotic closely related to cinnamycin (Ro09-0198), which is known to
Duramycin is a 19-amino-acid tetracyclic peptide produced
by Streptoverticillium cinnamoneus and is closely related to
cinnamycin (Ro09-0198) (Fig. 1 A) (1–5). Both compounds
characterized by the presence of a high proportion of unusual
phosphatidylethanolamine (PE) (6–8). Because of this char-
acteristic, cinnamycin has been employed to study the
distribution and metabolism of PE (9–14). Duramycin is
specificity of duramycin is not well established. Previously it
was proposed that duramycin recognizes a particular mem-
brane conformation determined by the presence of PE or
monogalactosyl diglyceride (MGDG) (15). Analysis of the
membranes of the duramycin-resistant Bacillus subtilis
mutants revealed that they had little or no PE and cardiolipin
(15,16). In contrast, mutation of alkalophilic Bacillus firmus
to duramycin resistance resulted in a substantial replacement
of PE by its plasmalogen form (17).
In eukaryotic cells, PE is mainly restricted to the inner
leaflet of the plasma membrane (19–21). Recently we showed
that cinnamycin induces transbilayer phospholipid move-
ment of target cells in a PE-dependent manner (8). This
causes exposure of the inner leaflet PE to the peptide and
promotes binding of cinnamycin. When the surface concen-
tration of PE is high, cinnamycin induces membrane reor-
ganization such as membrane fusion and the alteration of the
membrane gross morphology (8). However, the detailed
membrane ultrastructure induced by cinnamycin binding is
not well determined. Although duramycin was known to alter
the membrane permeability of mammalian cells (18,22,23),
the precise mechanism(s) of duramycin-induced membrane
damage is not yet determined.
cinnamycin with model membranes. The results indicate that
both duramycin and cinnamycin selectively bind ethanola-
mine phospholipids, irrespective of whether they are of
diacyl- or plasmalogen type. The binding of the lantibiotics
induces reorganization of the membrane into highly curved
tubular structures as revealed by electron microscopy and
small-angle x-ray scattering (SAXS). In addition, we found
the lantibiotics preferentially bind PE in the highly curved
membranes. Thus, both duramycin and cinnamycin promote
ment and by changing membrane curvature.
MATERIALS AND METHODS
The following were purchased from Avanti Polar Lipids (Alabaster, AL):
L-a-phosphatidylcholine (egg, chicken; egg PC).
L-a-phosphatidylethanolamine (egg, chicken; egg PE).
L-a-phosphatidylethanolamine (liver, bovine; liver PE).
Submitted November 21, 2006, and accepted for publication April 27, 2007.
Address reprint requests to Toshihide Kobayashi, Tel.: 81-48-467-9534;
Editor: Michael Edidin.
? 2007 by the Biophysical Society
1608 Biophysical JournalVolume 93 September 20071608–1619
1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 lyso-PE).
L-a-phosphatidylserine (brain, porcine; brain PS).
L-a-phosphatidylinositol (liver, bovine; liver PI).
L-phosphatidyl-DL-glycerol (egg, chicken; egg PG).
L-a-phosphatidic acid (egg, chicken; egg PA).
glycerol (18:1 BMP).
cardiolipin (heart, bovine; heart CL).
chicken; egg SM).
porcine; brain SM).
Total cerebrosides (brain, porcine; GalCer).
From Matreya (Pleasant Gap, PA):
Glucosylceramide (human; GlcCer), lactosylceramide (LacCer), and
monogalactosyl diglyceride (plant, hydrogenated; 18:0 MGDG).
From Larodan Fine Chemicals (Malmo ¨, Sweden):
Monogalactosyl diglyceride (plant MGDG).
From Wako Pure Chemical Industries (Osaka, Japan):
Ganglioside GM1(bovine brain; GM1), ganglioside GM2(NeuAc) (bovine
brain; GM2), and ganglioside GM3(NeuAc) (bovine; GM3).
From Sigma (St. Louis, MO):
1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyr-
istoyl-sn-glycero-3-phosphoethanolamine (DMPE), and duramycin and
Abu, a-aminobutyric acid; Ala, alanine; Asn, asparagine; Asp, aspartic acid; Gln, glutamine; Gly, glycine; Lys, lysine; Phe, phenylalanine; Pro, proline; Val,
valine. Ala6is linked to Lys19as lysinoalanine. Ala-S-Ala: lanthionine, Ala-S-Abu: b-methyllanthionine, X2: lysine (duramycin) or arginine (cinnamycin)
(4,5). (B) Rabbit erythrocytes (final 3 3 107cells/ml) were incubated with various concentrations of duramycin for 30 min at 4?C or 37?C. Hemolysis was
measured as described in Materials and Methods.(C) Duramycinwas preincubated with various concentrations of MLVs composed of POPC,90 mol % POPC
and 10 mol % SOPE, or 90 mol % POPC and 10 mol % C18(plasm)-18:1 PE for 1 h at 37?C. The mixtures (final concentration of duramycin was 5 mM) were
then added to rabbit erythrocytes (final 3 3 107cells/ml) and further incubated for 30 min at 37?C, followed by the measurement of hemolysis. Horizontal axis
indicates the final concentration of the total lipids in MLVs. (D) Duramycin was preincubated with MLVs containing 90 mol % POPC and 10 mol % of
indicated lipids, followed by the measurement of hemolysis, as described in panel C. Final concentrations of duramycin and total lipids were 5 mM and
500 mM, respectively. Data are means 6 SD of at least three independent experiments.
Ethanolamine phospholipids inhibit hemolytic activity of duramycin. (A) Structure of duramycin and cinnamycin (Ro09-0198). Abbreviations:
Curvature-Dependent PE-Binding Peptides 1609
Biophysical Journal 93(5) 1608–1619
surface of cinnamycin. Since the hydrophobic amino acids
are identical between cinnamycin and duramycin, one can
expect the penetration of duramycin to the hydrophobic
region of PE membrane.
Duramycin and cinnamycin promote membrane
binding by inducing transbilayer lipid movement
and by changing membrane curvature
PE mainly resides in the inner layer of the plasma membrane
(19,21,49). To induce cell lysis, duramycin and cinnamycin
the cell surface. Previously we showed that cinnamycin
promotes cell binding by inducing transbilayer lipid move-
ment (8). Transbilayer lipid movement in the plasma mem-
brane causes the exposure of PE to the outer leaflet. The
present study indicates that, in addition to inducing trans-
bilayer lipid movement, duramycin and cinnamycin alter
the membrane to highly curved tubular structures. Since
duramycin and cinnamycin prefer high curvature, the lanti-
biotics promote further binding of the peptides by inducing
tubulation. The mechanism of the lantibiotics-induced mem-
tubulation is accompanied by the transbilayer lipid move-
ment, and the resultant high curvature may be accounted for
by a biased outward-directed transbilayer lipid movement.
Exposure or high curvature?
Our results indicate that the binding of both duramycin and
cinnamycin to PE is dependent on the curvature of the mem-
tion of PE. It is reported that PE is exposed at restricted sites
of the cell surface. Using an amino-reactive probe, trinitro-
benzene sulfonic acid, it has been shown that in steady-state
fibroblasts, 2–2.5 mol % of total PE is exposed on the cell
surface (50,51). From the present results, one cannot exclude
the possibility that PE is evenly distributed on the cell sur-
face and cinnamycin recognizes the high curvature, instead
of the exposure of PE. Further studies are required to under-
stand cellular distribution of PE.
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
We are grateful to H. Iwase (Japan Atomic Energy Agency (JAEA)) and H.
Takahashi (Gunma University) for fruitful discussions on the analysis of
SAXS data, to A. Yamaji-Hasegawa (RIKEN) for the technical help in
measurement of hemolysis and helpful discussions, to T. Zimmer (Friedrich
Schiller Univ. Jena) for valuable discussions, to R. Ishitsuka, H.
Shogomori, Y. Ueda, K. Ishii, M. Abe, and K. Tamada for their help in
SAXS measurements at SPring-8, to K. Tamada, Y. Ueda, F. Hullin-
Matsuda, and R. Ishitsuka for critically reading the manuscript, and to all
members of the Kobayashi labs for valuable discussions.
This work was supported by grants from the Ministry of Education, Science,
Sports and Culture of Japan (Nos. 17390025 and 18050040 to T.K., No.
17659058 to M.M.), grants from RIKEN Frontier Research System, Chem-
ical Biology Project of RIKEN, RIKEN Presidential Research Grant for
Intersystem Collaboration (to T.K.), and a grant from the Hayashi Memorial
Foundation for Female Natural Scientists (to A.M.). K.I. is a Special
Postdoctoral Researcher of RIKEN.
1. Lindenfelser, L. A., T. G. Pridham, O. L. Shotwell, and F. H. Stodola.
1957. Antibiotics against plant disease. IV. Activity of duramycin
against selected microorganisms. Antibiot. Annu. 5:241–247.
2. Shotwell, O. L., and T. G. Pridham. 1958. Antibiotics against plant
disease. III. Duramycin, a new antibiotic from Streptomyces cinna-
moneus forma azacoluta. J. Am. Chem. Soc. 80:3912–3915.
3. Kessler, H., S. Steuernagel, and M. Will. 1988. The structure of the
polycyclic nonadecapeptide Ro 09–0198. Helv. Chim. Acta. 71:
4. Hayashi, F., K. Nagashima, Y. Terui, Y. Kawamura, K. Matsumoto,
and H. Itazaki. 1990. The structure of PA48009: the revised structure
of duramycin. J. Antibiot. (Tokyo). 43:1421–1430.
5. Fredenhagen, A., G. Fendrich, F. Marki, W. Marki, J. Gruner, F.
Raschdorf, and H. H. Peter. 1990. Duramycins B and C, two new
lanthionine containing antibiotics as inhibitors of phospholipase A2.
Structural revision of duramycin and cinnamycin. J. Antibiot. (Tokyo).
6. Choung, S. Y., T. Kobayashi, J. Inoue, K. Takemoto, H. Ishitsuka, and
K. Inoue. 1988. Hemolytic activity of a cyclic peptide Ro09–0198
isolated from Streptoverticillium. Biochim. Biophys. Acta. 940:171–179.
7. Choung, S. Y., T. Kobayashi, K. Takemoto, H. Ishitsuka, and K. Inoue.
1988. Interaction of a cyclic peptide, Ro09–0198, with phosphatidyl-
ethanolamine in liposomal membranes. Biochim. Biophys. Acta. 940:
8. Makino, A., T. Baba, K. Fujimoto, K. Iwamoto, Y. Yano, N. Terada, S.
Ohno, S. B. Sato, A. Ohta, M. Umeda, K. Matsuzaki, and T. Kobayashi.
2003. Cinnamycin (Ro 09–0198) promotes cell binding and toxicity by
inducing transbilayer lipid movement. J. Biol. Chem. 278:3204–3209.
9. Emoto, K., T. Kobayashi, A. Yamaji, H. Aizawa, I. Yahara, K. Inoue,
and M. Umeda. 1996. Redistribution of phosphatidylethanolamine at
the cleavage furrow of dividing cells during cytokinesis. Proc. Natl.
Acad. Sci. USA. 93:12867–12872.
10. Emoto, K., O. Kuge, M. Nishijima, and M. Umeda. 1999. Isolation
of a Chinese hamster ovary cell mutant defective in intramitochondrial
transport of phosphatidylserine. Proc. Natl. Acad. Sci. USA. 96:12400–
11. Emoto, K., and M. Umeda. 2000. An essential role for a membrane lipid
in cytokinesis. Regulation of contractile ring disassembly by redistribu-
tion of phosphatidylethanolamine. J. Cell Biol. 149:1215–1224.
12. Kato, U., K. Emoto, C. Fredriksson, H. Nakamura, A. Ohta, T.
Kobayashi, K. Murakami-Murofushi, and M. Umeda. 2002. A novel
membrane protein, Ros3p, is required for phospholipid translocation
across the plasma membrane in Saccharomyces cerevisiae. J. Biol.
13. Iwamoto, K., S. Kobayashi, R. Fukuda, M. Umeda, T. Kobayashi, and
A. Ohta. 2004. Local exposure of phosphatidylethanolamine on the
yeast plasma membrane is implicated in cell polarity. Genes Cells. 9:
14. Emoto, K., H. Inadome, Y. Kanaho, S. Narumiya, and M. Umeda.
2005. Local change in phospholipid composition at the cleavage fur-
row is essential for completion of cytokinesis. J. Biol. Chem. 280:
15. Navarro, J., J. Chabot, K. Sherrill, R. Aneja, S. A. Zahler, and E.
Racker. 1985. Interaction of duramycin with artificial and natural mem-
branes. Biochemistry. 24:4645–4650.
1618 Iwamoto et al.
Biophysical Journal 93(5) 1608–1619
16. Dunkley, E. A., Jr., S. Clejan, A. A. Guffanti, and T. A. Krulwich.
1988. Large decreases in membrane phosphatidylethanolamine and
diphosphatidylglycerol upon mutation to duramycin resistance do not
change the protonophore resistance of Bacillus subtilis. Biochim.
Biophys. Acta. 943:13–18.
17. Clejan, S., A. A. Guffanti, M. A. Cohen, and T. A. Krulwich. 1989.
Mutation of Bacillus firmus OF4 to duramycin resistance results in
substantial replacement of membrane lipid phosphatidylethanolamine
by its plasmalogen form. J. Bacteriol. 171:1744–1746.
channel formation by duramycin. Biochim. Biophys. Acta. 1107:179–185.
19. Devaux, P. F. 1991. Static and dynamic lipid asymmetry in cell mem-
branes. Biochemistry. 30:1163–1173.
20. Cerbon, J., and V. Calderon. 1991. Changes of the compositional
asymmetry of phospholipids associated to the increment in the
membrane surface potential. Biochim. Biophys. Acta. 1067:139–144.
21. Zachowski, A. 1993. Phospholipids in animal eukaryotic membranes:
transverse asymmetry and movement. Biochem. J. 294:1–14.
22. Racker, E., C. Riegler, and M. Abdel-Ghany. 1984. Stimulation of
glycolysis by placental polypeptides and inhibition by duramycin.
Cancer Res. 44:1364–1367.
23. Roberts, M., S. B. Hladky, R. J. Pickles, and A. W. Cuthbert. 1991.
Stimulation of sodium transport by duramycin in cultured human
colonic epithelia. J. Pharmacol. Exp. Ther. 259:1050–1058.
25. Yamaji, A., Y. Sekizawa, K. Emoto, H. Sakuraba, K. Inoue, H.
Kobayashi, and M. Umeda. 1998. Lysenin, a novel sphingomyelin-
specific binding protein. J. Biol. Chem. 273:5300–5306.
26. Rouser, G., A. N. Siakotos, and S. Fleisher. 1966. Quantitative analysis
of phospholipids by thin-layer chromatography and phosphorus anal-
ysis of spots. Lipids. 1:85–86.
27. Ishitsuka, R., A. Yamaji-Hasegawa, A. Makino, Y. Hirabayashi, and
T. Kobayashi. 2004. A lipid-specific toxin reveals heterogeneity of
sphingomyelin-containing membranes. Biophys. J. 86:296–307.
28. Reference deleted in proof.
29. Chonn, A., S. C. Semple, and P. R. Cullis. 1991. Separation of large
unilamellar liposomes from blood components by a spin column
procedure: towards identifying plasma proteins which mediate lipo-
some clearance in vivo. Biochim. Biophys. Acta. 1070:215–222.
30. Ishitsuka, R., and T. Kobayashi. 2007. Cholesterol and lipid/protein
ratio control the oligomerization of a sphingomyelin-specific toxin,
lysenin. Biochemistry. 46:1495–1502.
31. Fujisawa, T., K. Inoue, T. Oka, H. Iwamoto, T. Uraga, T. Kumasaka,
Y. Inoko, N. Yagi, M. Yamamoto, and T. Ueki. 2000. Small-angle x-ray
scattering station at the SPring-8 RIKEN beamline. J. Appl. Crystallogr.
32. Fujisawa, T., Y. Nishikawa, H. Yamazaki, and Y. Inoko. 2003.
Evaluation and improvements of the Rigaku imaging plate reader
(R-Axis IV11) for the use in synchrotron x-ray solution scattering.
J. Appl. Crystallogr. 36:535–539.
33. Reference deleted in proof.
34. Huang, T., H. Toraya, T. Blanton, and Y. Wu. 1993. X-ray powder
diffraction analysis of silver behenate, a possible low-angle diffraction
standard. J. Appl. Crystallogr. 26:180–184.
35. Glatter, O., and O. Kratky. 1982. Small Angle X-Ray Scattering.
Academic Press, London.
36. Guinier, A., and G. Fournet. 1955. Small Angle Scattering Of X-Rays.
Wiley, New York.
37. Wakamatsu, K., S. Y. Choung, T. Kobayashi, K. Inoue, T.
Higashijima, and T. Miyazawa. 1990. Complex formation of peptide
antibiotic Ro09–0198 with lysophosphatidylethanolamine: 1H NMR
analyses in dimethyl sulfoxide solution. Biochemistry. 29:113–118.
38. Machaidze, G., A. Ziegler, and J. Seelig. 2002. Specific binding of Ro
09–0198 (cinnamycin) to phosphatidylethanolamine: a thermodynamic
analysis. Biochemistry. 41:1965–1971.
39. Marsh, D. 1990. CRC Handbook of Lipid Bilayers. CRC Press, Boca
40. Machaidze, G., and J. Seelig. 2003. Specific binding of cinnamycin
(Ro 09–0198) to phosphatidylethanolamine. Comparison between
micellar and membrane environments. Biochemistry. 42:12570–12576.
41. Pabst, G., M. Rappolt, H. Amenitsch, and P. Laggner. 2000. Structural
information from multilamellar liposomes at full hydration: full q-range
fitting with high quality x-ray data. Phys. Rev. E Stat. Phys. Plasmas
Fluids Relat. Interdiscip. Topics. 62:4000–4009.
42. Rappolt, M., A. Hickel, F. Bringezu, and K. Lohner. 2003. Mechanism
of the lamellar/inverse hexagonal phase transition examined by high
resolution x-ray diffraction. Biophys. J. 84:3111–3122.
43. Kucerka, N., S. Tristram-Nagle, and J. F. Nagle. 2005. Structure of
fully hydrated fluid phase lipid bilayers with monounsaturated chains.
J. Membr. Biol. 208:193–202.
44. Zamyatnin, A. A. 1972. Protein volume in solution. Prog. Biophys.
Mol. Biol. 24:107–123.
45. Perkins, J. P. 1988. Modern Physical Methods in Biochemistry, Part B. A.
Neuberger and L. L. M. van Deenen, editors. Elsevier, Amsterdam, The
46. Hirai, M., H. Iwase, T. Hayakawa, M. Koizumi, and H. Takahashi. 2003.
Determination of asymmetric structure of ganglioside-DPPC mixed
vesicle using SANS, SAXS, and DLS. Biophys. J. 85:1600–1610.
47. Balgavy, P., M. Dubnickova, N. Kucerka, M. A. Kiselev, S. P.
Yaradaikin, and D. Uhrikova. 2001. Bilayer thickness and lipid inter-
face area in unilamellar extruded 1,2-diacylphosphatidylcholine lipo-
somes: a small-angle neutron scattering study. Biochim. Biophys. Acta.
48. Nagle, J. F., and S. Tristram-Nagle. 2000. Structure of lipid bilayers.
Biochim. Biophys. Acta. 1469:159–195.
49. Cerbon, J., and V. Calderon. 1995. Generation, modulation and
maintenance of the plasma membrane asymmetric phospholipid com-
position in yeast cells during growth: their relation to surface poten-
tial and membrane protein activity. Biochim. Biophys. Acta. 1235:
50. Sleight, R. G., and R. E. Pagano. 1983. Rapid appearance of newly
synthesized phosphatidylethanolamine at the plasma membrane. J. Biol.
51. Kobayashi, T., and R. E. Pagano. 1989. Lipid transport during mitosis.
Alternative pathways for delivery of newly synthesized lipids to the
cell surface. J. Biol. Chem. 264:5966–5973.
52. Rappolt, M., K. Pressl, G. Pabst, and P. Laggner. 1998. La-phase
separation in phosphatidylcholine-water systems induced by alkali
chlorides. Biochim. Biophys. Acta. 1372:389–393.
Curvature-Dependent PE-Binding Peptides1619
Biophysical Journal 93(5) 1608–1619