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W1988127877 abstract "Recently two β-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053–51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel β-sheet stabilized by three disulfide bridges with a pairing typical of β-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian β-defensins showed that the hydrophobic patch is not preserved in mammalian β-defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids. Recently two β-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053–51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel β-sheet stabilized by three disulfide bridges with a pairing typical of β-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian β-defensins showed that the hydrophobic patch is not preserved in mammalian β-defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids. During the final stage of egg incubation in king penguins (Aptenodytes patagonicus), the male can preserve undigested food in the stomach for several weeks (1Gauthier-Clerc M. Le Maho Y. Clerquin Y. Drault S. Handrich Y. Nature. 2000; 408: 928-929Crossref PubMed Scopus (40) Google Scholar). This ensures survival of the newly hatched chick in the event that the return of the foraging female from the sea is delayed. In accordance with the characterization of stress-induced bacteria (2Thouzeau C. Froget G. Monteil H. Le Maho Y. Harf-Monteil C. Polar Biol. 2003; 26: 115-123Crossref Scopus (12) Google Scholar), a previous study has demonstrated that numerous antimicrobial activities exist in preserved stomach contents (3Thouzeau C. Le Maho Y. Froget G. Sabatier L. Le Bohec C. Hoffmann J.A. Bulet P. J. Biol. Chem. 2003; 278: 51053-51058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Two antimicrobial peptides have been isolated and fully characterized, namely spheniscin-1 and -2 (Sphe-1/pBD-1 and Sphe-2/pBD-2). 1The abbreviations used are: Sphe-1/pBD-1 and Sphe-2/pBD-2, spheniscin-1/penguin β-defensin-1 and spheniscin-2/penguin β-defensin-2, respectively; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; r.m.s.d., root mean square deviation; Gal, gallinacin; CHP, chicken heterophil peptide; GPV, gallopavin; THP, turkey heterophil peptide; HBD, human β-defensin; mBD, murine β-defensin; BNBD, bovine neutrophil β-defensin; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MIC, minimal inhibitory concentration; RK, rabbit kidney; PDB, Protein Data Bank.1The abbreviations used are: Sphe-1/pBD-1 and Sphe-2/pBD-2, spheniscin-1/penguin β-defensin-1 and spheniscin-2/penguin β-defensin-2, respectively; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; r.m.s.d., root mean square deviation; Gal, gallinacin; CHP, chicken heterophil peptide; GPV, gallopavin; THP, turkey heterophil peptide; HBD, human β-defensin; mBD, murine β-defensin; BNBD, bovine neutrophil β-defensin; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MIC, minimal inhibitory concentration; RK, rabbit kidney; PDB, Protein Data Bank. The two forms of spheniscins differ by a single residue, His14, in Sphe-1 versus Arg14 in Sphe-2. A data bank search revealed that spheniscins are members of the well known defensin family. Defensins are small (3–5 kDa) cationic antimicrobial peptides that are part of the innate immunity of vertebrates (4Ganz T. Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2319) Google Scholar, 5Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6696) Google Scholar), invertebrates (6Bulet P. Charlet M. Hetru C. Ezekowitz R. Innate Immunity. Humana Press Inc., Totowa, NJ2002: 89-107Google Scholar), and plants (7Garcia-Olmedo F. Molina A. Alamillo J.M. Rodriguez-Palenzuela P. Biopolymers. 1998; 47: 479-491Crossref PubMed Scopus (458) Google Scholar). In vertebrates, defensins can be divided into α-, β-, and θ-defensin subfamilies. The unusual θ-defensin, which is a circular peptide, was initially isolated from rhesus macaque (8Tang Y.Q. Yuan J. Osapay G. Osapay K. Tran D. Miller C.J. Ouellette A.J. Selsted M.E. Science. 1999; 286: 498-502Crossref PubMed Scopus (616) Google Scholar). Recently a pseudogene coding for a homologue, named retrocyclin, was found in the human genome (9Cole A.M. Hong T. Boo L.M. Nguyen T. Zhao C. Bristol G. Zack J.A. Waring A.J. Yang O.O. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1813-1818Crossref PubMed Scopus (265) Google Scholar). The α- and β-defensins are the most widespread in nature, and they are subclassified according to precursor and gene structures and to the position and connectivity of the six cysteine residues of their sequence. In α-defensins, the disulfide pairing is Cys1-Cys6, Cys2-Cys4, and Cys3-Cys5, whereas it is Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6 in β-defensins. In spheniscins, the disulfide pattern is identical to that of β-defensins (3Thouzeau C. Le Maho Y. Froget G. Sabatier L. Le Bohec C. Hoffmann J.A. Bulet P. J. Biol. Chem. 2003; 278: 51053-51058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). β-Defensins are more widely distributed than α-defensins as only β-defensins have been isolated from birds. In addition to the two spheniscins isolated from the king penguin, bird β-defensins have been found in polymorphonucleated granulocytes and in epithelia of chickens and turkeys. Four β-defensins named gallinacins and chicken heterophil peptides (Gal-1/CHP1, Gal-1α/CHP2, Gal-2, and Gal-3) have been reported in Gallus gallus (10Harwig S.S. Swiderek K.M. Kokryakov V.N. Tan L. Lee T.D. Panyutich E.A. Aleshina G.M. Shamova O.V. Lehrer R.I. FEBS Lett. 1994; 342: 281-285Crossref PubMed Scopus (180) Google Scholar, 11Evans E.W. Beach G.G. Wunderlich J. Harmon B.G. J. Leukoc. Biol. 1994; 56: 661-665Crossref PubMed Scopus (149) Google Scholar, 12Zhao C. Nguyen T. Liu L. Sacco R.E. Brogden K.A. Lehrer R.I. Infect. Immun. 2001; 69: 2684-2691Crossref PubMed Scopus (142) Google Scholar), and four have been reported in the turkey Meleagris gallopavo, named gallopavin-1 (GPV-1) and THP-1, THP-2, and THP-3 for turkey heterophil peptides 1–3 (11Evans E.W. Beach G.G. Wunderlich J. Harmon B.G. J. Leukoc. Biol. 1994; 56: 661-665Crossref PubMed Scopus (149) Google Scholar, 12Zhao C. Nguyen T. Liu L. Sacco R.E. Brogden K.A. Lehrer R.I. Infect. Immun. 2001; 69: 2684-2691Crossref PubMed Scopus (142) Google Scholar). Sphe-1 and -2 have been isolated from the stomach contents of king penguins that efficiently conserve food for several weeks (3Thouzeau C. Le Maho Y. Froget G. Sabatier L. Le Bohec C. Hoffmann J.A. Bulet P. J. Biol. Chem. 2003; 278: 51053-51058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The activity of a synthetic version of Sphe-2 is preferentially directed against Gram-positive bacteria including pathogenic strains and is of bactericidal type. Interestingly, when an activity was recorded against Gram-negative strains, the effect of Sphe-2 is mainly of bacteriostatic type. This contrasts with the activity generally observed for β-defensins as most of them are effective against Gram-negative bacteria and yeast cells. In fact, the activity of spheniscin is closer to that of α-defensins that are microbicidal against Gram-positive and Gram-negative bacteria, yeast fungi, and several enveloped viruses (4Ganz T. Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2319) Google Scholar). This biological property of the spheniscins compared with other vertebrate β-defensins in addition to (i) the low percentage of similarity with mammalian β-defensins, (ii) their high efficacy on human pathogenic fungi, and (iii) their location within the rather unfavorable environment of the penguin stomach prompted us to investigate the three-dimensional structure of Sphe-2. The structure of six β-defensins has been solved by NMR or x-ray crystallography (13Schibli D.J. Hunter H.N. Aseyev V. Starner T.D. Wiencek J.M. McCray Jr., P.B. Tack B.F. Vogel H.J. J. Biol. Chem. 2002; 277: 8279-8289Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 14Mandal M. Jagannadham M.V. Nagaraj R. Peptides. 2002; 23: 413-418Crossref PubMed Scopus (55) Google Scholar, 15Bauer F. Schweimer K. Kluver E. Conejo-Garcia J.R. Forssmann W.G. Rosch P. Adermann K. Sticht H. Protein Sci. 2001; 10: 2470-2479Crossref PubMed Scopus (148) Google Scholar, 16Sawai M.V. Jia H.P. Liu L. Aseyev V. Wiencek J.M. McCray Jr., P.B. Ganz T. Kearney W.R. Tack B.F. Biochemistry. 2001; 40: 3810-3816Crossref PubMed Scopus (110) Google Scholar, 17Hoover D.M. Chertov O. Lubkowski J. J. Biol. Chem. 2001; 276: 39021-39026Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 18Hoover D.M. Rajashankar K.R. Blumenthal R. Puri A. Oppenheim J.J. Chertov O. Lubkowski J. J. Biol. Chem. 2000; 275: 32911-32918Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 19Zimmermann G.R. Legault P. Selsted M.E. Pardi A. Biochemistry. 1995; 34: 13663-13671Crossref PubMed Scopus (143) Google Scholar): human β-defensins HBD-1, HBD-2, and HBD-3; murine β-defensins mBD-7 and mBD-8; and bovine neutrophil β-defensin-12 (BNBD-12), but no crystal or NMR solution structure of a bird β-defensin is available. In this study, we determined the three-dimensional structure of the king penguin β-defensin, Sphe-2, in aqueous solution by two-dimensional 1H NMR spectroscopy and molecular modeling and compared this structure to those of the closest β-defensins from mammals. The global fold of Sphe-2, as of most β-defensins, includes a well defined three-stranded β-sheet and a short N-terminal domain with an α-helical propensity. However, differences were observed in the distribution of the charged and hydrophobic residues. While HBD-2 and BNBD-12 are amphipathic, HBD-3 is mainly hydrophilic. Sphe-2, although very cationic, is slightly less hydrophilic than HBD-3, and its surface displays a small hydrophobic patch that was not described in HBD-3 but apparently preserved in avian β-defensins according to comparative modeling studies. These results provide evidence that penguin β-defensin has some structural peculiarities that may explain the differences in the antimicrobial spectrum and activities between the bird and the mammalian β-defensins. Peptide Synthesis and Purification—Synthetic Sphe-2 was obtained from Altergen Laboratory (Schiltigheim, France). The integrity, purity, and correct refolding were confirmed by MALDI-TOF-MS fingerprinting of a tryptic digest of 2 μg of peptide following the experimental procedure reported previously (3Thouzeau C. Le Maho Y. Froget G. Sabatier L. Le Bohec C. Hoffmann J.A. Bulet P. J. Biol. Chem. 2003; 278: 51053-51058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Briefly purified Sphe-2 (native and synthetic) was treated with trypsin (Roche Applied Science) at an enzyme/substrate ratio of 1:10 (w/w), and the digest was analyzed by MALDI-TOF-MS using α-cyano-4-hydroxycinnamic acid as matrix. NMR Experiments—The NMR sample was prepared by dissolving 5.5 mg of synthetic Sphe-2 in 600 μlofH2O/D2O (90:10) to obtain a final concentration of 2 mm. All 1H NMR spectra were recorded on a Varian INOVA NMR spectrometer equipped with a z-axis field-gradient unit and operating at a proton frequency of 600 MHz. The pH was adjusted to 4.3, and the double quantum filtered correlation spectroscopy (20Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2596) Google Scholar), clean TOCSY (21Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1193) Google Scholar), and NOESY (22Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4828) Google Scholar) experiments were performed at 293K. Clean TOCSY was acquired using a mixing time of 80 ms; NOESY spectra were performed with mixing times of 120, 160, and 300 ms. All spectra were referenced to the residual H2O signal set as the carrier frequency, 4.821 ppm at 293 K, and were processed with NMRPipe/NMRDraw softwares (23Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11450) Google Scholar). The identification of amino acid spin systems and the sequential assignment were performed using the standard strategy described by Wüthrich (24Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley Interscience, New York1986: 130-161Google Scholar). To identify the exchange rate of backbone amide protons, the sample was lyophilized and quickly dissolved in D2O. The exchange kinetic experiments were then monitored from one-dimensional spectra at different time intervals and from short TOCSY spectra. Structure Calculations—Distance constraints were determined by volume integration of correlations observed in the NOESY spectrum recorded with a mixing time of 160 ms using the NMRView software (25Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2670) Google Scholar). Since the disulfide pairing is known (3Thouzeau C. Le Maho Y. Froget G. Sabatier L. Le Bohec C. Hoffmann J.A. Bulet P. J. Biol. Chem. 2003; 278: 51053-51058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), covalent bonds were built between the sulfur atoms of the paired cysteine residues, Cys5-Cys33, Cys12-Cys27, and Cys17-Cys34. Structure calculations were carried out with ARIA 1.1 (26Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (332) Google Scholar) implemented in the software CNS 1.1 (27Brünger A.T. Adams P.D. Clore G.M. De Lano W.L. Gros P. Grosse-Kuntsleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). ARIA is a powerful approach using an iterative process to perform the assignment of ambiguous NOEs from structures calculated using a combination of NOEs assigned at the previous step and a set of ambiguous distance restraints treated as the sum of contributions from all possible assignments. Each ARIA run included nine iterations. In the last step, the 20 best structures were refined by molecular dynamics in explicit solvent to remove artifacts due to the simplification of the force field used in the previous steps. A table of chemical shifts and a first set of distance restraints deduced from easily assigned NOEs were introduced as input to ARIA, and the calculations were initiated. The “ambicutoff” p (26Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (332) Google Scholar) for accepting or rejecting possible assignments was modified compared with the default values. In the standard ARIA protocol, p varies from 0.999 to 0.8 during the whole process. The variation was reduced from 0.999 to 0.9 to prevent the elimination of weak contributions to ambiguous cross-peaks that are essential for the formation of the secondary structure elements. At the end of each run, the new assignments proposed by ARIA were checked manually and introduced (or not) in the following run. Rejected restraints and residual violations were also analyzed, and assignments were corrected if required. This iterative process was repeated until complete assignment of the NOESY map was achieved. A last run of 100 structures was then performed with the final list of NOE-derived distance restraints in which no restraint can be rejected. The 10 structures with the lowest energy were considered as characteristic of the peptide structure. The structures were displayed and analyzed using the MOLMOL (28Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6477) Google Scholar) and SYBYL (SYBYL®6.5, TRIPOS Inc., St. Louis, MO) programs, and their quality was evaluated using the PROCHECK (29Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4363) Google Scholar) and PROMOTIF (30Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (994) Google Scholar) softwares. The formation of hydrogen bonds was established according to distance criteria. Lipophilic and electrostatic potentials were calculated and represented using the MOLCAD option of SYBYL. Salt Effect on the Antibacterial Activity of Sphe-2—The effect of sodium and magnesium chloride on the antibacterial activity of Sphe-2 was assessed using a liquid growth inhibition assay (31Hetru C. Bulet P. Methods Mol. Biol. 1997; 78: 35-49PubMed Google Scholar). Growth inhibition was estimated by measurement of the minimal inhibitory concentration (MIC) against selected strains, the Gram-negative Escherichia coli and the pathogenic Gram-positive Staphylococcus aureus. Briefly logarithmic phase bacterial cultures were diluted to an A600 of 0.001 (approximately 105 colony-forming units/ml) in a broth supplemented or not with various concentration of salts. Diluted bacteria (90 μl) were mixed with 10 μl of either distilled water (control) or different concentrations of Sphe-2 ranging from 0.75 up to 100 μm. Bacterial growth was measured after an overnight incubation at 30 °C by measuring the change in the absorbance of the culture at 600 nm with a microplate reader. The MIC value corresponds to the interval of concentration [a]–[b] where [a] is the highest concentration tested at which the bacteria are growing and [b] is the lowest concentration that causes 100% inhibitory growth (32Casteels P. Tempst P. Biochem. Biophys. Res. Commun. 1994; 199: 339-345Crossref PubMed Scopus (132) Google Scholar). To assay the salt effect on the bacterial growth, the poor broth medium (1% bactotryptone, 85 mm NaCl, pH 7.4) was supplemented with NaCl and MgCl2 separately or in combination. The following final salt concentrations were assayed: (i) 160 and 480 mm in NaCl, (ii) 1 and 50 mm in MgCl2, and (iii) 160 mm NaCl plus 1 mm MgCl2. Osmolarity of the different media and of the stomach content was determined using an automatic milliosmometer (type 13/13DR, Roebling, Berlin, Germany). NMR Data—After identification of the spin systems on the correlation spectroscopy and TOCSY spectra, sequence-specific assignments were obtained from the sequential connectivities observed on the amide-α, amide-amide cross-peak region of the NOESY spectrum. The fingerprint region of the TOCSY and NOESY spectra are shown in Fig. 1 (see the complete maps in the supporting information). All protons were assigned except for the amide proton of Phe2, which was not observed in our experimental conditions; the arginine ηNH2; the serine OH; and the N-terminal NH +3 groups, which are in very fast exchange with water. In addition, the ξ protons of Phe residues could not be assigned unambiguously due to overlapping with the other aromatic protons (see the chemical shift table in the supporting information). Low field shifted Hα chemical shifts and a characteristic set of interstrand Hα(i)-Hα(j), NH(i)-NH(j), and Hα(i)-NH(j) connectivities delineate a triple-stranded β-sheet. The presence of medium range NOEs in the rest of the sequence suggests that it consists mainly of loops and turns. Strong sequential Hα/Hδ NOE cross-peaks for Xaa-Pro peptide bonds indicate a trans configuration for Pro20 and Pro23. Twenty-one NH signals, exchanged before the acquisition of the first one-dimensional spectra (t = 5 min), are described as “very fast exchanging protons.” Four additional amide proton signals disappeared within the first 18 h. The residual amide protons, not exchanged after 18 h, are all located in the β-sheet (Phe11, Ala13, Ile22, Ile24, Cys27, Gln32, Cys33, Cys34, and Arg35) except for the NH of Gly10 and Val31 (Fig. 1) (see the short TOCSY spectra recorded in D2O to monitor the exchange data in the supporting information). Structure Calculations—The NOE data set used in the final ARIA run included 594 distance restraints (Table I) involving 335.2 intraresidue, 139.7 sequential, 32 medium range (2 ≤ |i–j| ≤ 4), and 87.1 long range (|i–j| ≥ 5) restraints with an average of 16 restraints/residue. Among these restraints, 551 are non-ambiguous. Ten structures, in very good agreement with all the experimental data and the standard covalent geometry, were selected for further analysis. For these structures, no experimental distance constraint violation greater than 0.3 Å was observed and the root mean square deviation (r.m.s.d.) values, with respect to the standard geometry, are low. The Ramachandran plot exhibits 95% of the ϕ, ψ angles of the 10 converged structures in the most favored and additional allowed regions according to the PROCHECK software nomenclature. The selected structures display small potential energy values. In particular negative van der Waals and electrostatic energy values are indicative of favorable non-bonded interactions.Table IStructural statistics for the 10 final models of Sphe-2NOE restraintsTotal594Intraresidue (|i - j| = 0)335.2Sequential (|i - j| = 1)139.7Medium range (2 ≤|i - j| ≤ 4)32.0Long range (|i - j| ≥ 5)87.1Ambiguous restraints43r.m.s.d. on backbone Cα atoms (pairwise, Å)Global (residues 2-37)1.93 ± 0.45Triple-stranded β-sheet (residues 11-13, 21-27, and 31-36)0.54 ± 0.10β1 (residues 11-13)0.10 ± 0.07β2 (residues 21-27)0.45 ± 0.12β3 (residues 31-36)0.36 ± 0.12N terminus (residues 2-10)2.40 ± 0.73L1 (residues 14-20)1.55 ± 0.62Ramachandran plotaDetermined by PROCHECK. (%)Most favored regions71.7Additional allowed regions23.3Generously allowed regions3.3Disallowed regions1.7EnergiesbCalculated with the standard parameters of ARIA. (kcal·mol-1)Electrostatic-930 ± 55van der Waals-129 ± 9ENOE9 ± 3Total energy-827 ± 68a Determined by PROCHECK.b Calculated with the standard parameters of ARIA. Open table in a new tab Structure Description—The overall fold of Sphe-2 is typical of β-defensins including a twisted three-stranded antiparallel β-sheet with a (+2X, –1) topology as defined by Richardson (33Richardson J.S. Anfinsen C.B. Edsall J.T. Richard F.M. Advances in Protein Chemistry. 34. Academic Press, New York1981: 167-339Google Scholar). Strand β1 (Phe11–Ala13) is hydrogen-bonded to strand β3 (Val31–Arg36), which in turn is hydrogen-bonded to strand β2 (Ser21–Cys27) (Fig. 2). Residues Ile24 and Gly25 are both hydrogen-bonded to Cys33 (Fig. 1) leading to a β-bulge in β2. The amide protons of all residues involved in hydrogen bonds exhibited a slow exchange rate in D2O (Fig. 1), and the two other residues with a slow NH exchange rate, Gly10 and Val31, are embedded in the structure and particularly inaccessible to the solvent. Strands β1 and β2 are separated by a long loop, L1, including a type IV β-turn between residues Arg18 and Ser21 and a type I β-turn involving residues Ser28 to Val31. Due to a low number of NOEs in this region, the N-terminal segment (Ser1–Gly10) is less “well” defined (Fig. 2). In fact, a small helical turn is formed on a majority of structures, but depending on the structure it was considered by PROMOTIF as an α-helix or as a succession of turns. In addition, a PROCHECK analysis showed that most residues in the segment from Phe2 to Arg8 lie in the helical region of the Ramachandran plot. As evidenced by the r.m.s.d. calculations (Table I) and by a superposition of the backbone of the selected structures (Fig. 2), the β-sheet region of Sphe-2 is well defined with a pairwise r.m.s.d. on the Cα atoms of 0.54 Å. The pairwise r.m.s.d. calculated for all Cα backbone atoms is large (1.93 Å) due to the N-terminal segment (2.40 Å) and to the L1 loop (1.55 Å). The tertiary structure of Sphe-2 is very compact. The hydrogen bonds between the three strands of the β-sheet and the three disulfide bridges contribute to the compactness of this molecule. Most residues are exposed to the solvent and exhibit a large variability in the position of their side chains. The only residues showing a low circular variance of their χ1, χ2 angles are hydrophobic ones, Phe11, Phe19, Ile22, Ile24, Phe30, and Val31. Several of these residues contribute to the formation of a hydrophobic pole at the surface of the molecule: Phe19, Pro20, Ile22, Val37, and Trp38 (Fig. 3A). The other hydrophobic residues are scattered all over the rest of the molecule and are separated by hydrophilic residues. With 10 arginine residues over 38 amino acids and no anionic residue, Sphe-2 is a highly cationic molecule. The charges are spread all over the surface of the molecule, and the electrostatic potential calculated with SYBYL using Kollman charges (34Pearlman D.A. Case D.A. Calwell J.W. Ross W.S. Cheatham T.E. DeBold S. Ferguson D. Seibel G. Kollman P. Comp. Phys. Commun. 1995; 91: 1-4Crossref Scopus (2647) Google Scholar) varied between –15 and +380 kcal·mol–1 so that the surface of the molecule is almost entirely positively charged except for the C terminus (Fig. 3A). Salt Sensitivity of Sphe-2—In control poor broth medium (85 mm NaCl, 216 mosm), Sphe-2 is active at a concentration below 6 μm against the two microorganisms selected for this study, S. aureus (Gram-positive) and E. coli (Gram-negative) (Table II). An increase in the NaCl content to a concentration of 160 mm, representing an intermediate concentration between the values observed in seawater fishes and mammals (35Eckert R. Randall D. Augustine G. Animal Physiology. Mechanisms and Adaptations. W. H. Freeman and Co., New York1988: 389Google Scholar), did not affect Sphe-2 activity. The osmolarity measured for this culture medium (348 mosm) was close to the value measured in the stomach content of three different penguins (324 ± 23 mosm) and to the osmolarity of the plasma of sea water fishes (337 mosm) (35Eckert R. Randall D. Augustine G. Animal Physiology. Mechanisms and Adaptations. W. H. Freeman and Co., New York1988: 389Google Scholar). When the concentration in sodium chloride was increased up to 480 mm, the efficacy of Sphe-2 against S. aureus decreased by a factor of 16 (MIC, 50–100 μm). In the control experiment with poor broth supplemented at 480 mm NaCl, E. coli did not grow properly.Table IIEffect of sodium and magnesium chloride on Sphe-2 activityFinal salt concentration in poor brothOsmolarityMICS. aureusE. colimosmμm85 mm NaClaControl poor broth.2163-61.5-3.0160 mm NaCl3483-61.5-3.0480 mm NaCl96150-100—bThe bacterial strain did not grow properly in the control experiment.85 mm NaCl + 1 mm MgCl22203-63-685 mm NaCl + 50 mm MgCl234425-506-12cAbnormal bacterial growth, characterized by the presence of a bacterial pellet, was present.160 mm NaCl + 1 mm MgCl23526-123-6a Control poor broth.b The bacterial strain did not grow properly in the control experiment.c Abnormal bacterial growth, characterized by the presen" @default.
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W1988127877 title "Solution Structure of Spheniscin, a β-Defensin from the Penguin Stomach" @default.
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W1988127877 doi "https://doi.org/10.1074/jbc.m401338200" @default.
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