The Three-dimensional Structure of Carnocyclin A Reveals That Many Circular Bacteriocins Share a Common Structural Motif

Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes. Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes. Bacteriocins are a diverse group of ribosomally synthesized, antimicrobial peptides produced by bacteria. Those made by Gram-positive bacteria are usually cationic and typically have 30–70 residues (1Cotter P.D. Hill C. Ross R.P. Nat. Rev. Microbiol. 2005; 3: 777-788Crossref PubMed Scopus (1665) Google Scholar, 2Nes I.F. Yoon S.S. Diep W.B. Food Sci. Biotechnol. 2007; 16: 675-690Google Scholar, 3van Belkum M.J. Stiles M.E. Nat. Prod. Rep. 2000; 17: 323-335Crossref PubMed Scopus (91) Google Scholar). These peptides are substantially more active than conventional antibiotics against numerous pathogenic and drug resistant bacteria, including virulent strains of Staphylococci, Enterococci, Listeria, and Clostridia, but they display virtually no toxicity toward eukaryotic cells. The circular bacteriocins are a unique group, characterized by an amide bond linking the N and C termini of the peptide. They exhibit enhanced stability to pH and temperature variation and are resistant to numerous proteases, in contrast to many linear bacteriocins. This stability derives, in part, from the cyclic structure of the peptide (4Maqueda M. Sánchez-Hidalgo M. Fernández M. Montalbán-López M. Valdivia E. Martínez-Bueno M. FEMS Microbiol. Rev. 2008; 32: 2-22Crossref PubMed Scopus (128) Google Scholar). Interestingly, circular peptides are not unique to bacteria; they have also been discovered in plants and animals and exhibit a diverse range of bioactivities. Typically, the circular peptides from these higher organisms are shorter in length and contain at least one disulfide bond, further bracing the structure and enhancing stability (5Craik D.J. Science. 2006; 311: 1563-1564Crossref PubMed Scopus (241) Google Scholar, 6Craik D.J. Daly N.L. Saska I. Trabi M. Rosengren K.J. J. Bacteriol. 2003; 185: 4011-4021Crossref PubMed Scopus (47) Google Scholar). We recently isolated carnocyclin A (CclA) 2The abbreviations used are: CclAcarnocyclin Aαα-helixMES2-(N-morpholine)ethanesulfonic acidAS-48enterocin AS-48NOEnuclear Overhauser enhancementHPLChigh performance liquid chromatographyTAPS[(2-hydroxy-1,1-bis[hydroxymethyl]ethyl)amino]-1-propanesulfonic acidr.m.s.d.root mean square deviationHSQCheteronuclear single quantum coherenceTOCSYtotal correlation spectroscopyCOSYcorrelation spectroscopyNOESYNOE spectroscopy. from Carnobacterium maltaromaticum UAL307 and employed tandem mass spectrometry amino acid sequencing and genetic analysis to confirm that it is a circular bacteriocin (7Martin-Visscher L.A. van Belkum M.J. Garneau-Tsodikova S. Whittal R.M. Zheng J. McMullen L.M. Vederas J.C. Appl. Environ. Microbiol. 2008; 74: 4756-4763Crossref PubMed Scopus (118) Google Scholar). This 60-residue peptide displays a broad antimicrobial spectrum and is particularly potent against the serious food pathogen Listeria monocytogenes. The producer strain has recently been approved in the United States as an additive for preservation of processed meat products. carnocyclin A α-helix 2-(N-morpholine)ethanesulfonic acid enterocin AS-48 nuclear Overhauser enhancement high performance liquid chromatography [(2-hydroxy-1,1-bis[hydroxymethyl]ethyl)amino]-1-propanesulfonic acid root mean square deviation heteronuclear single quantum coherence total correlation spectroscopy correlation spectroscopy NOE spectroscopy. Eight other circular bacteriocins have been reported. These include enterocin AS-48 (8Samyn B. Martinez-Bueno M. Devreese B. Maqueda M. Gálvez A. Valdivia E. Coyette J. Van Beeumen J. FEBS Lett. 1994; 352: 87-90Crossref PubMed Scopus (87) Google Scholar), butyrivbriocin AR10 (9Kalmokoff M.L. Cyr T.D. Hefford M.A. Whitford M.F. Teather R.M. Can. J. Microbiol. 2003; 49: 763-773Crossref PubMed Scopus (51) Google Scholar), circularin A (10Kemperman R. Kuipers A. Karsens H. Nauta A. Kuipers O. Kok J. Appl. Environ. Microbiol. 2003; 69: 1589-1597Crossref PubMed Scopus (116) Google Scholar), gassericin A and reutericin 6 (11Kawai Y. Ishii Y. Arakawa K. Uemura K. Saitoh B. Nishimura J. Kitazawa H. Yamazaki Y. Tateno Y. Itoh T. Saito T. Appl. Environ. Microbiol. 2004; 70: 2906-2911Crossref PubMed Scopus (76) Google Scholar, 12Kawai Y. Saito T. Kitazawa H. Itoh T. Biosci. Biotechnol. Biochem. 1998; 62: 2438-2440Crossref PubMed Scopus (81) Google Scholar), subtilosin A (13Kawulka K. Sprules T. McKay R.T. Mercier P. Diaper C.M. Zuber P. Vederas J.C. J. Am. Chem. Soc. 2003; 125: 4726-4727Crossref PubMed Scopus (101) Google Scholar, 14Babasaki K. Takao T. Shimonishi Y. Kurahashi K. J. Biochem. 1985; 98: 585-603Crossref PubMed Scopus (182) Google Scholar), uberloysin (15Wirawan R.E. Swanson K.M. Kleffmann T. Jack R.W. Tagg J.R. Microbiology. 2007; 153: 1619-1630Crossref PubMed Scopus (92) Google Scholar), and most recently, lactocyclicin Q (16Sawa N. Zendo T. Kiyofuji J. Fujita K. Himeno K. Nakayama J. Sonomoto K. Appl. Envir. Microbiol. 2009; 75: 1552-1558Crossref PubMed Scopus (95) Google Scholar). Gassericin A and reutericin 6 have identical primary sequences but differ by the presence of one d-alanine, resulting in different spectra of activity and secondary structure profiles (11Kawai Y. Ishii Y. Arakawa K. Uemura K. Saitoh B. Nishimura J. Kitazawa H. Yamazaki Y. Tateno Y. Itoh T. Saito T. Appl. Environ. Microbiol. 2004; 70: 2906-2911Crossref PubMed Scopus (76) Google Scholar). Acidocin B is considered a putative circular protein, as it shows 98% sequence identity to gassericin A and reutericin 6, but its circular nature has not been confirmed (17Leer R.J. van der Vossen J.M.B.M. van Giezen M. van Noort J.M. Pouwels P.H. Microbiology. 1995; 141: 1629-1635Crossref PubMed Scopus (106) Google Scholar). Of these bacteriocins, subtilosin A is atypical; it is significantly shorter (35 amino acids), anionic, and contains unique thioether bridges linking cysteine sulfurs to the α-carbon of other residues (13Kawulka K. Sprules T. McKay R.T. Mercier P. Diaper C.M. Zuber P. Vederas J.C. J. Am. Chem. Soc. 2003; 125: 4726-4727Crossref PubMed Scopus (101) Google Scholar, 18Kawulka K.E. Sprules T. Diaper C.M. Whittal R.M. McKay R.T. Mercier P. Zuber P. Vederas J.C. Biochemistry. 2004; 43: 3385-3395Crossref PubMed Scopus (167) Google Scholar, 19Zheng G. Yan L.Z. Vederas J.C. Zuber P. J. Bacteriol. 1999; 181: 7346-7355Crossref PubMed Google Scholar). As such, subtilosin A represents a unique class of bacteriocins (13Kawulka K. Sprules T. McKay R.T. Mercier P. Diaper C.M. Zuber P. Vederas J.C. J. Am. Chem. Soc. 2003; 125: 4726-4727Crossref PubMed Scopus (101) Google Scholar) and will not be included in the present discussion of the other circular bacteriocins. These range from 58–70 amino acids in length, are cationic, and contain a large number of hydrophobic residues (4Maqueda M. Sánchez-Hidalgo M. Fernández M. Montalbán-López M. Valdivia E. Martínez-Bueno M. FEMS Microbiol. Rev. 2008; 32: 2-22Crossref PubMed Scopus (128) Google Scholar). To date, the structure of only one of these circular bacteriocins has been in reported. In 2000, González et al. (20González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11221-11226Crossref PubMed Scopus (154) Google Scholar) described the NMR solution structure of AS-48, revealing that it consists of five helices encompassing a compact hydrophobic core. The covalent bond linking the N and C termini of the peptide was found to reside within the fifth helix. In 2003, crystallographic studies supported the proposal that at AS-48 as a in the hydrophobic of the are in and with the are M.J. M. Gálvez A. Valdivia E. Maqueda M. A. J. 2003; PubMed Scopus Google Scholar). with a this a its hydrophobic and into the The three-dimensional structure of AS-48 shows a the as a of at one of the a high of the of the peptide. This is to be for of the peptide into the membrane a as (20González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11221-11226Crossref PubMed Scopus (154) Google Scholar, M.A. Valdivia E. Maqueda M. Rico M. J. Sci. 2005; PubMed Scopus Google Scholar, M. Gálvez A. M.J. González C. A. Rico M. Valdivia E. Sci. 2004; PubMed Scopus Google Scholar). studies of AS-48 have that this peptide in lipid for the of and low the membrane A. Maqueda M. Martínez-Bueno M. Valdivia E. J. Bacteriol. PubMed Google Scholar). A similar of has been for gassericin A and reutericin 6 (11Kawai Y. Ishii Y. Arakawa K. Uemura K. Saitoh B. Nishimura J. Kitazawa H. Yamazaki Y. Tateno Y. Itoh T. Saito T. Appl. Environ. Microbiol. 2004; 70: 2906-2911Crossref PubMed Scopus (76) Google Scholar). We have now determined the three-dimensional solution structure of CclA by Our results reveal that CclA a globular of four helices surrounding a hydrophobic core. The of CclA and its are similar to of AS-48, a common structural for circular bacteriocins that resembles the a structural found within the and E. M. J.M. G. Nat. PubMed Scopus Google Scholar, M. M. M. K. H. Biochemistry. 2006; PubMed Scopus Google Scholar). studies show that CclA forms anion selective in lipid bilayers L.A. Vederas J.C. M. 2009; PubMed Scopus Google Scholar). We have further characterized these ion by the anion of the and the of pH channel NMR studies that CclA binds fluoride in two different The results show that despite the shared structural of a circular bacteriocins have unique properties. CclA was from C. maltaromaticum UAL307 as described (7Martin-Visscher L.A. van Belkum M.J. Garneau-Tsodikova S. Whittal R.M. Zheng J. McMullen L.M. Vederas J.C. Appl. Environ. Microbiol. 2008; 74: 4756-4763Crossref PubMed Scopus (118) Google Scholar). NMR CclA was as a by the producer in of was identical to that of the was in in and to a of To and acid was for NMR spectra a with an and the the was and in an of and the and and from the and processed with S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and with J. 1994; PubMed Scopus Google Scholar). The structure of CclA was with P. C. K. J. PubMed Scopus Google Scholar), nuclear Overhauser from the and and and from analysis of the and from G. A. J. 1999; PubMed Scopus Google Scholar). A of and used and within to of a total of NOE and used in the The with no NOE of and no residues in the of the as of the solution structure of CclA. for CclA have been in the and have been in the with S. M.J. Proc. Natl. Acad. Sci. U.S.A. 98: PubMed Scopus Google Scholar), and The secondary structure of the circular bacteriocins was with the C. 2008; PubMed Scopus Google Scholar) and K. 2000; 16: PubMed Scopus Google Scholar, J. 1999; PubMed Scopus Google Scholar) structure To for the of secondary structure the N and C sequence was as In the the linear sequence was and from the The sequence was that a the N and the residues to the C of the This sequence was for the sequences of AS-48, circularin and lactocyclicin Q with M.A. G. R. H. A. R. 2007; PubMed Scopus Google Scholar). The sequences into N. PubMed Scopus Google Scholar) and the structure of either AS-48 or to the The to the for further N. PubMed Scopus Google Scholar). bilayers and CclA as described L.A. Vederas J.C. M. 2009; PubMed Scopus Google Scholar). CclA to the and single channel was as anion single channel The and the was or with pH the solution was from pH to either for pH or to for pH pH the solution was used in the and for the the pH CclA was in and to a of The pH of the was to acid The was with fluoride to pH by of and of The resulting spectra by NMR a with a of with fluoride and acid in the of also at pH to NMR a analysis of the amino acids in CclA was as two of the circular bacteriocins A and reutericin are to contain (11Kawai Y. Ishii Y. Arakawa K. Uemura K. Saitoh B. Nishimura J. Kitazawa H. Yamazaki Y. Tateno Y. Itoh T. Saito T. Appl. Environ. Microbiol. 2004; 70: 2906-2911Crossref PubMed Scopus (76) Google Scholar). and chromatography mass it was found that that CclA contains only acids are in the To the oligomeric of CclA different pH and a of and NMR Our results show in contrast to AS-48, CclA is in To NMR a of CclA was by the producer in The spectrum of CclA shows with of the unique A of of the of CclA O. in O. Scholar). analysis J. 1994; PubMed Scopus Google Scholar) of the that CclA contains four by not In that the N and C termini of CclA are in a a of and heteronuclear The structure of CclA was with P. C. K. J. PubMed Scopus Google Scholar), a of and from NOE and from the and the G. A. J. 1999; PubMed Scopus Google Scholar). A of of the solution structure of was This of is as in The structural for this of are in CclA contains four comprised of residues and The the N and C termini of CclA is within In the helices are by a more is helices and This to contains and the of in the these residues are from the the of the from to The four helices of CclA are with of hydrophobic into the of the Several range hydrophobic the arrangement of the four for range range range and with residues in most in in in and and with residues in a of CclA. the hydrophobic of CclA. are as hydrophobic from and of CclA. and and residues are The show the of most of the hydrophobic of CclA are in the hydrophobic are hydrophobic residues These residues a hydrophobic that the of and The structure of AS-48 similar as the of a from hydrophobic residues the of and M.J. M. Gálvez A. Valdivia E. Maqueda M. A. J. 2003; PubMed Scopus Google Scholar). As results show that CclA not in these hydrophobic of the peptide with the of the CclA also has a hydrophobic the and This may be for and with the for the of the N and C termini of CclA. The of CclA the of a four sequence and the covalent of two within The of is by this as such, the hydrophobic nature of the may of the linear with the the termini of the peptide into and the hydrophobic residues that In to hydrophobic the of CclA a and CclA contains five four of are within this and a similar is with AS-48, reside in this (20González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11221-11226Crossref PubMed Scopus (154) Google Scholar). In the of AS-48, it has been that this is for membrane a as (20González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11221-11226Crossref PubMed Scopus (154) Google Scholar, M. Gálvez A. M.J. González C. A. Rico M. Valdivia E. Sci. 2004; PubMed Scopus Google Scholar). to this a peptide binds to the of a the with the peptide of the for of the peptide M. M. A. G. FEBS Lett. 1999; PubMed Scopus Google Scholar). it has also been that that this is not for In et al. M.A. Valdivia E. Maqueda M. Rico M. J. Sci. 2005; PubMed Scopus Google Scholar) that a of AS-48, these and a similar to the not exhibit As such, this of the peptide in with other of the bacteriocin to the bacteria. The structure of CclA is similar to that of AS-48, sequences only are globular comprised of four or five helices a hydrophobic core. The two surprisingly with an of the sequence The of CclA and AS-48 shows that in the helices are similar in length and similar and are as are of CclA and of In the covalent the N and C termini in the at the three-dimensional In to a similar CclA many with AS-48, particularly the hydrophobic and cationic The most CclA and AS-48 is that the contains an helix. of that of AS-48 and the of CclA the CclA is shorter than AS-48 by it is to a at this be of CclA and of In of CclA is and has a and its not with of As be from the the and in the shorter CclA to its with The S. P. A. 2008; PubMed Scopus Google Scholar) was used to with similar to CclA. As the was with AS-48 and In and peptides also A and C P. J.R. Sci. 2006; PubMed Scopus (76) Google Scholar), as lipid and are to with the found to be similar to CclA and and despite low sequence In the peptides A O. van K. H. M. J. J. Chem. 2004; PubMed Scopus Google Scholar) and E. M. J.M. G. Nat. PubMed Scopus Google Scholar), lyse and eukaryotic also high structural to CclA and and The sequence and structural of CclA to these and peptides is in has been that the circular bacteriocins be into two to sequence (1Cotter P.D. Hill C. Ross R.P. Nat. Rev. Microbiol. 2005; 3: 777-788Crossref PubMed Scopus (1665) Google Scholar). contains the disparate bacteriocins lactocyclicin AS-48, circularin and the bacteriocins reutericin 6, and group Our results reveal that CclA and AS-48 have low sequence identity, three-dimensional are studies of gassericin A and reutericin 6 and sequence analysis of lactocyclicin Q that these bacteriocins also contain secondary structure (11Kawai Y. Ishii Y. Arakawa K. Uemura K. Saitoh B. Nishimura J. Kitazawa H. Yamazaki Y. Tateno Y. Itoh T. Saito T. Appl. Environ. Microbiol. 2004; 70: 2906-2911Crossref PubMed Scopus (76) Google Scholar, N. Zendo T. Kiyofuji J. Fujita K. Himeno K. Nakayama J. Sonomoto K. Appl. Envir. Microbiol. 2009; 75: 1552-1558Crossref PubMed Scopus (95) Google Scholar). We that of circular bacteriocins the common structural of a fold E. M. J.M. G. Nat. PubMed Scopus Google Scholar). To examined a of secondary structure and and the group circular bacteriocins, sequence shows that the the of these and this is to contain two from residues and CclA and lactocyclicin a of or residues with a high number of residues and is found by a this the structure of CclA in a from and a the bacteriocins AS-48, circularin and an is As been from the sequence and the of CclA with AS-48, this is it is likely that circularin A and contain five helices the shorter circular bacteriocins have four To further these of the group bacteriocins. Q was circularin A and also the structure of CclA by it The resulting of CclA to the NMR solution with in the of the four The structural and of CclA and AS-48 also the of the other bacteriocins of group these bacteriocins have a with hydrophobic In they exhibit in different of the primary sequences shows that AS-48, and circularin A have a high of residues toward the of for CclA and for AS-48 and circularin the sequence of shows residues in the N and C termini Q from the other bacteriocins in this residues are more the sequence of this with only a at the The of lactocyclicin Q also shows the of this bacteriocin the bacteriocins of reutericin 6, and sequence analysis four in length, residues and They are by either or This group, although to low to the group bacteriocins. the of this are as these peptides contain many more residues and have that of it likely that the structure of this group of bacteriocins that of with four helices a compact hydrophobic with an studies have that CclA forms anion selective in lipid bilayers L.A. Vederas J.C. M. 2009; PubMed Scopus Google Scholar). We have further characterized the of these by a of anion either halide anions and or anions and the single channel was within the range to and was to and The from the membrane the and to the sequence J.M. Rev. PubMed Scopus Google Scholar). This that the an anion and a channel binding is than the to the anion of J.M. Rev. PubMed Scopus Google Scholar). We also examined the of pH the of the CclA pores. different pH the for the CclA linear and channel the was to be and at pH and The was in the was at pH and the was at pH These results suggest that the pH of the solution not channel as was at of the pH This is in with NMR studies that show in of CclA a broad range of pH pH not channel it has a the stability of the CclA is that the CclA ion channel is more and that anion is the five of CclA be imparting a the the of also be further enhancing the of the This is likely a in CclA to the lipid membrane and membrane M. M. A. G. FEBS Lett. 1999; PubMed Scopus Google Scholar). A similar has been in the of by AS-48 (20González C. Langdon G.M. Bruix M. Gálvez A. Valdivia E. Maqueda M. Rico M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11221-11226Crossref PubMed Scopus (154) Google Scholar, M. Gálvez A. M.J. González C. A. Rico M. Valdivia E. Sci. 2004; PubMed Scopus Google Scholar). AS-48, by CclA is the membrane CclA channel and with and with This the of to and is than the voltage-dependent in ion as and B. of Scholar). CclA channel is by membrane the was and at The membrane no CclA channel in the range of to not studies that CclA is of fluoride examined the fluoride and CclA by In an of CclA was with and the of the fluoride was by The results reveal at least two modes of binding fluoride and CclA to the of a at is This fluoride likely from to has been to of fluoride ion in solution S.S. J. P. J. Am. Chem. Soc. 2009; PubMed Scopus Google Scholar). CclA of fluoride and at of fluoride and a at This has a to that for fluoride anion in the of CclA and represents the results at least two different binding modes CclA and low of fluoride one of this is by as the of this anion and a the fluoride and CclA be by the of fluoride the and of CclA was examined by NMR spectroscopy. of no in the of CclA in the an of the five and the identity of these was with a spectrum are in the no in the spectra of these fluoride was This is not as CclA binds and different including and CclA contains as its from the be by a large at in the NMR to of the residues in fluoride binding are likely in an to with and no structural of CclA the of other anions and the structure of CclA not in by the results suggest that the two different by NMR for fluoride to CclA may be to different and of to this anion at a single In studies show that despite sequence the circular bacteriocins likely a common structural motif. This consists of four or five helices a compact hydrophobic with the common of the The bacteriocins with primary sequences are to the is This unique stability to these potent antimicrobial peptides and modes of at the The of CclA and AS-48 and the of circularin and lactocyclicin Q show that a of residues a the of the peptide. This is likely for the peptides to the of the in as anion by CclA and by AS-48, most likely from of the structural and studies the circular bacteriocins will these peptides We Sprules McKay of and for in NMR spectra and to of of for We and Mercier for with and and of of for with with

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