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• W1971891360 abstract "Defensins are small cationic peptides that are crucial components of innate immunity, serving as both antimicrobial agents and chemoattractant molecules. The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect. Here, we report two high resolution x-ray structures of human β-defensin-2 (hBD2). The phases were experimentally determined by the multiwavelength anomalous diffraction method, utilizing a novel, rapid method of derivatization with halide ions. Although the shape and charge distribution of the monomer are similar to those of other defensins, an additional α-helical region makes this protein topologically distinct from the mammalian α- and β-defensin structures reported previously. hBD2 forms dimers topologically distinct from that of human neutrophil peptide-3. The quaternary octameric arrangement of hBD2 is conserved in two crystal forms. These structures provide the first detailed description of dimerization of β-defensins, and we postulate that the mode of dimerization of hBD2 is representative of other β-defensins. The structural and electrostatic properties of the hBD2 octamer support an electrostatic charge-based mechanism of membrane permeabilization by β-defensins, rather than a mechanism based on formation of bilayer-spanning pores. Defensins are small cationic peptides that are crucial components of innate immunity, serving as both antimicrobial agents and chemoattractant molecules. The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect. Here, we report two high resolution x-ray structures of human β-defensin-2 (hBD2). The phases were experimentally determined by the multiwavelength anomalous diffraction method, utilizing a novel, rapid method of derivatization with halide ions. Although the shape and charge distribution of the monomer are similar to those of other defensins, an additional α-helical region makes this protein topologically distinct from the mammalian α- and β-defensin structures reported previously. hBD2 forms dimers topologically distinct from that of human neutrophil peptide-3. The quaternary octameric arrangement of hBD2 is conserved in two crystal forms. These structures provide the first detailed description of dimerization of β-defensins, and we postulate that the mode of dimerization of hBD2 is representative of other β-defensins. The structural and electrostatic properties of the hBD2 octamer support an electrostatic charge-based mechanism of membrane permeabilization by β-defensins, rather than a mechanism based on formation of bilayer-spanning pores. human β -defensin asymmetric unit(s) bovine β-defensin human neutrophil peptide multiwavelength anomalous diffraction nuclear magnetic resonance polyethylene glycol 4-morpholinepropanesulfonic acid Multicellular organisms share an innate defense against microorganisms that is based on small cationic peptides known as defensins (1Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1998; 10: 41-44Crossref PubMed Scopus (339) Google Scholar, 2White S.H. Wimley W.C. Selsted M.E. Curr. Opin. Struct. Biol. 1995; 5: 521-527Crossref PubMed Scopus (382) Google Scholar). Mammals contain two classes of defensins, named α and β, based on the arrangement of cysteines within their sequences. To date, six α-defensins and two β-defensins (hBD11 and hBD2) have been identified in humans. Four of the six α-defensins are sequestered in secretory granules within neutrophils and are termed neutrophil peptides (hNP1–4), whereas the remaining two α-defensins (human α-defensins 5 and 6) are secreted from Paneth cells in the gastrointestinal tract. α-Defensins manifest microbicidal activity only at relatively high concentrations and have a broad spectrum of activity, killing Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses (3Lehrer R.I. Ganz T. Ann. N. Y. Acad. Sci. 1996; 797: 228-239Crossref PubMed Scopus (116) Google Scholar). In contrast, β-defensins are inducible, more potent, and selective, killing mainly Gram-negative bacteria and yeast (4Harder J. Bartels J. Christophers E. Schröder J.M. Nature. 1997; 387: 861Crossref PubMed Scopus (1194) Google Scholar). Both α- and β-defensins also mobilize cells engaged in adaptive immune responses (5Chertov O. Michiel D.F. Xu L. Wang J.M. Tani K. Murphy W.J. Longo D.L. Taub D.D. Oppenheim J.J. J. Biol. Chem. 1996; 271: 2935-2940Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 6Yang D. Chertov O. Bykovskaia S.N. Chen Q. Buffo M.J. Shogan J. Anderson M. Schroder J.M. Wang J.M. Howard O.M. Oppenheim J.J. Science. 1999; 286: 525-528Crossref PubMed Scopus (1538) Google Scholar). The microbicidal activity of defensins stems from the permeabilization of anionic lipid bilayers and the subsequent release of cellular contents (7Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (590) Google Scholar, 8Viljanen P. Koski P. Vaara M. Infect. Immun. 1988; 56: 2324-2329Crossref PubMed Google Scholar, 9Rana F. Blazyk J. Prog. Clin. Biol. Res. 1989; 292: 77-85PubMed Google Scholar, 10Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar, 11Fujii G. Selsted M.E. Eisenberg D. Protein Sci. 1993; 2: 1301-1312Crossref PubMed Scopus (148) Google Scholar). Interactions between defensins and bacterial membranes are governed mainly by electrostatic forces (11Fujii G. Selsted M.E. Eisenberg D. Protein Sci. 1993; 2: 1301-1312Crossref PubMed Scopus (148) Google Scholar). One mechanism of permeabilization is thought to involve the formation of ion pores in bacterial membranes (12Kagan B.L. Selsted M.E. Ganz T. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 210-214Crossref PubMed Scopus (451) Google Scholar, 13Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar). The existence of pores and the estimation of their dimensions are based on studies of ion conductance through the bilayer (14Cociancich S. Ghazi A. Hetru C. Hoffmann J.A. Letellier L. J. Biol. Chem. 1993; 268: 19239-19245Abstract Full Text PDF PubMed Google Scholar, 15Gálvez A. Maqueda M. Martı́nez-Bueno M. Valdivia E. J. Bacteriol. 1991; 173: 886-892Crossref PubMed Google Scholar) and the passage of molecules of various sizes through model lipid vesicles and bilayers (13Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 16Hristova K. Selsted M.E. White S.H. J. Biol. Chem. 1997; 272: 24224-24233Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A physical model of such a pore has been constructed based on the x-ray structure of hNP3 (11Fujii G. Selsted M.E. Eisenberg D. Protein Sci. 1993; 2: 1301-1312Crossref PubMed Scopus (148) Google Scholar, 13Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 17Hill C.P. Yee J. Selsted M.E. Eisenberg D. Science. 1991; 251: 1481-1485Crossref PubMed Scopus (456) Google Scholar). Based on its amphiphilic charge distribution and dimeric shape, 12 monomers of hNP3 were arranged to form a membrane-spanning pore of ∼20 Å inner diameter (13Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 16Hristova K. Selsted M.E. White S.H. J. Biol. Chem. 1997; 272: 24224-24233Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A second model has also been proposed for other small cationic antimicrobial peptides, such as magainins (18Ludtke S.J. He K. Heller W.T. Harroun T.A. Yang L. Huang H.W. Biochemistry. 1996; 35: 13723-13728Crossref PubMed Scopus (694) Google Scholar) and cecropins (19Bechinger B. J. Membr. Biol. 1997; 156: 197-211Crossref PubMed Scopus (553) Google Scholar). According to this model (sometimes called the “carpet” model), the structural aspects of the interaction between peptides and the microbial membrane are of secondary importance. Rather than forming distinct and structurally stable ion pores, these peptides are thought to aggregate into positively charged patches that neutralize anionic lipid headgroups of the membrane over a wide area around the peptides. This neutralization disrupts the integrity of the lipid bilayer, causing transient gaps to arise and allowing ions (and larger molecules, depending on the extent of local disruption) to permeate the membrane (20Shai Y. Biochim. Biophys. Acta. 1999; 1462: 55-70Crossref PubMed Scopus (1591) Google Scholar). Except for the x-ray structure of hNP3, no other oligomeric structures of defensins have been determined. Because oligomerization of defensins may occur only at elevated protein concentrations (>3 mm) and possibly in the sterically constraining environment of a bacterial membrane, a crystalline environment is probably suitable for studying the quaternary structure of defensins. To investigate the importance of the structure for the antimicrobial activity of hBD2, we solved the x-ray structure of this defensin by using a novel method of derivatizing the protein crystals, followed by the multiwavelength anomalous diffraction (MAD) phasing protocol. Additionally, we studied the permeabilization of large unilamellar vesicles by native and reductively alkylated hBD2, as well as the oligomerization of native hBD2 in solution. Protein was obtained from PeproTech (Rocky Hill, NJ). The homogeneity of the preparation was determined by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. Crystals were obtained by hanging-drop diffusion with equal volumes of concentrated protein (30 mg/ml after dissolving lyophilized protein in water) and reservoir solution containing 30% PEG 4000, 0.1 m Tris-HCl (pH 8.5), 0.2 mLi2SO4. Two crystal forms grew together in the same hanging drops. The orthorhombic form belongs to space group P21212 with cell constants a = 50.05 Å, b = 103.91 Å, and c = 28.27 Å. The monoclinic form belongs to space group P21 with cell constants a = 54.53 Å, b = 79.95 Å, c = 74.27 Å, and β = 105.30°. Individual crystal forms could be identified by their morphologies, with the orthorhombic form growing as rods and the monoclinic forms growing as square plates. The crystal forms were propagated and grown by macroseeding hanging drops preequilibrated to 20% PEG 4000, 0.066m Tris-HCl (pH 8.5), 0.133 mLi2SO4 with washed seed crystals of the intended form. The monoclinic form was the preferred form. Data were collected using flash-frozen crystals. Prior to freezing in the 100 K nitrogen stream, crystals were soaked in cryoprotectant solution containing 36% PEG 4000, 0.16 m MOPS (pH 7.1), 0.32m Li2SO4, 10% glycerol for approximately 60 s. Native and derivative data of both crystal forms were collected at beamline X9B (National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY) using an ADSC Quantum 4 CCD detector. CCD images were indexed, processed, merged, and scaled using DENZO and SCALEPACK (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The data collection statistics are shown in Table I.Table IData and refinement statisticsData setMonoclinicOrthorhombicBr1 (peak)Br2 (inflect.)Br3 (remote)IWavelength (Å)0.9790.9790.919820.920230.913451.542Resolution range (Å)30–1.730–1.3525–1.425–1.425–2.030–1.9Space groupP21P21212P21212P21212P21212P21212Unit cell (Å)a = 54.53a = 50.05a = 49.18a = 49.05b = 79.95b = 103.91b = 102.85b = 103.33c = 74.27c = 28.27c = 27.94c = 28.03β = 105.30Measured reflections165,583137,572123,991123,22450,88373,458Unique reflections65,64432,22728,74328,95910,17611,529Completeness 1-aNumbers in parentheses represent values for only those reflections within the highest-resolution shell, as determined using DENZO (21). (%)97.1 (96.3)96.7 (90.1)99.7 (98.2)98.4 (86.0)99.9 (100)97.3 (94.8)R merge a,b (%)4.3 (27.3)3.1 (20.4)3.4 (32.0)3.2 (33.3)3.1 (7.7)7.5 (30.3)I/ςI1-aNumbers in parentheses represent values for only those reflections within the highest-resolution shell, as determined using DENZO (21).24.2 (4.7)55.0 (6.5)53.6 (4.0)47.9 (4.3)42.6 (16.8)23.6 (6.5)Refinement R free,R work 1-cRwork = ∑‖{‖F o(h)‖ −k‖F o(h)‖}‖/∑‖F o(h)‖;R free = ∑(h)εT‖{‖F o(h)‖ −k‖F c(h)‖}‖/∑(h)εT‖F o(h)‖, where T represents a test set of reflections (10% of total, chosen at random) not used in the refinement.26.4, 18.823.7, 16.4 No. of atoms5,8181,416 No. of residues656162 No. of waters814194 Root mean square deviation Bond length (Å)0.0060.011 Bond angles (°)1.2301.932 Mean Bvalue Overall (Å2)29.329.6 Protein (Å2)28.928.9 Solvent (Å2)39.045.0b Rmerge = ∑‖I n − 〈I〉‖/∑〈I〉.1-a Numbers in parentheses represent values for only those reflections within the highest-resolution shell, as determined using DENZO (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar).1-c Rwork = ∑‖{‖F o(h)‖ −k‖F o(h)‖}‖/∑‖F o(h)‖;R free = ∑(h)εT‖{‖F o(h)‖ −k‖F c(h)‖}‖/∑(h)εT‖F o(h)‖, where T represents a test set of reflections (10% of total, chosen at random) not used in the refinement. Open table in a new tab b Rmerge = ∑‖I n − 〈I〉‖/∑〈I〉. Orthorhombic form crystals were soaked briefly (60 s) against KBr and KI (0.25 m) in the cryoprotectant solution immediately prior to freezing in the nitrogen stream and diffraction experiments (22Dauter Z. Dauter M. Rajashankar K.R. Acta Crystallogr. D. 2000; 56: 232-237Crossref PubMed Scopus (285) Google Scholar). MAD data at three wavelengths (see Table I) were collected for the KBr-soaked crystal, and one data set was collected at 1.54 Å for the KI-soaked crystal. The anomalous differences calculated by PHASES (23Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar) were 2.1% (based on F 2) and 1.5% (based on F) (1.4-Å resolution) for the KBr-soaked crystal data set, and 4.1% (based on F 2) and 2.7% (based onF) (1.9-Å resolution) for the KI-soaked crystal. Bromide and iodide sites were located from the anomalous differences using SHELXS (24Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-344Crossref PubMed Scopus (1892) Google Scholar). The highest nine E-map peaks from the KBr peak wavelength anomalous data were seen to be almost identical with those from the KI anomalous data, and so the same set of sites was used in refinement of the anomalous scatterer sites and phase generation. Anomalous scatterer sites were refined for each of the four data sets, and initial phases were generated for data from 24- to 2.0-Å resolution using MLPHARE (25Otwinowski Z. Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, Warrington, United Kingdom1991Google Scholar). The phases were then extended to 1.4 Å and refined with a solvent content of 35% using DM (26Cowtan K. Joint CCP4 ESF-EACBM Newsl. Protein Crystallogr. 1994; 31: 34-38Google Scholar). Because the handedness of the calculated phases was unknown, both the original positions of the sites (x, y,z) and their negative inversions (−x, −y, −z) were refined. Although the resulting figures of merit for both coordinate sets were indistinguishable, the rate of convergence to a final solution for the correct set of coordinates using DM was significantly faster compared with its enantiomorph. The final map from phase refinement and solvent flattening was easily interpretable. An initial model was built automatically using the program ARP/wARP (27Lamzin V.S. Wilson K.S. Methods Enzymol. 1997; 277: 269-305Crossref PubMed Scopus (278) Google Scholar) by placing single atoms (oxygen atoms) into electron density peaks. The protein model was then automatically built based on the positions of the oxygen atoms, and the structure factors were improved by combining the initial experimental phases with those calculated from the model. In total, 1,334 atoms were built into the map (out of a total 1,424, or ∼94% of the final model). The orthorhombic form model was completed using O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refined to 1.35 Å using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve 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. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), followed by SHELXL (24Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-344Crossref PubMed Scopus (1892) Google Scholar). In the final steps, anisotropic B factors for all atoms were individually refined, and 194 water molecules and one sulfate ion were added. TheR value for all reflections (20–1.35 Å) is 15.9% (R free 23.5%). The solution of the monoclinic form was obtained by molecular replacement technique using AMoRe (30Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar). Based on the estimated solvent content and strong noncrystallographic 422 symmetry seen in Patterson self-rotations of the monomeric crystal form, 16 independent monomers were expected to reside within the a.u. Because of the conservation of dimeric interfaces between the AB and CD dimers within the orthorhombic crystal form, a dimer containing chains A and B of the orthorhombic form was used as the search model. Searches using various resolution ranges were only successful in locating six of the eight dimers within the a.u. The best results were obtained for the resolution range 15–3.8 Å. The remaining two dimers were located on inspection of the resulting ‖Fo ‖ − ‖Fc ‖ maps, and by applying noncrystallographic transformations onto those chains already found. The 16 monomers of the monoclinic model were positioned as rigid bodies, then refined to 1.7-Å resolution in cycles of least-squares minimization and B factor refinement using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve 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. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Water molecules were placed automatically with a density cutoff of 2.5 ς and hydrogen bonding constraints using the program SHELXL (24Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-344Crossref PubMed Scopus (1892) Google Scholar), and visually inspected after completion of structural refinement. The final monoclinic model contains 814 water molecules and 5 sulfate ions were added. The R value for all reflections (25–1.7 Å) is 18.8% (Rfree 25.5%). hBD2 was dissolved in 100 mm Tris (pH 8.0) to 30 mg/ml. The protein was cleared by centrifugation and filtered using a 0.05-μm filter immediately prior to light-scattering measurements. A DynaPro-801 instrument was used for the measurements, and multicomponent analysis was performed using the software provided with the instrument. Large unilamellar vesicles were formed by extrusion through 0.2-μm nucleopore polycarbonate membranes under nitrogen pressure as described (13Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar) and loaded with 5,6-carboxyfluorescein (376 Da) fluorescein dextran (3 or 10 kDa). All reagents were from Molecular Probes (Eugene, OR). The reduction and carboxyamidomethylation of hBD2 were performed as described (31Walker J.M. The Protein Protocols Handbook. Humana Press, Totowa, NJ1996Crossref Google Scholar). Mass spectrometry analysis of modified hBD2 revealed alkylation of all six cysteine residues. To show that the activity of hBD2 is dependent not on charge alone but also on its native structure, we performed experiments on the permeabilization of artificial lipid vesicles, using both native and reduced hBD2. During our experiments, native hBD2 (0.125 μm) induced a 50% release of a small (400 Da) marker from large unilamellar vesicles made from anionic lipid palmitoyloleoylphosphatidyglycerol. Under these conditions, about 20% of 3-kDa dextran and less than 5% of 10-kDa dextran were released. The effect of hBD2 was lipid dependent; the increase in the proportion of neutral palmitoyloleoylphosphatidylcholine led to a dramatically decreased sensitivity to hBD2, indicating the electrostatic nature of hBD2 interaction with lipids. The correct fold of hBD2 is clearly required for this effect, because hBD2 linearized by disulfide bond reduction and carboxyamidomethylation was ineffective even at concentrations that resulted in 100% release of the vesicle contents induced by native hBD2. These results indicate that both the native structure of hBD2 and its charge interaction with the membrane are critical for the membrane permeabilizing activity of this protein. The crystal structure of the orthorhombic form was solved by the MAD technique, using an anomalous signal originating from bromide and iodide anions soaked into the crystals of the hBD2 derivatives. The method that we used to derivatize the protein has been successfully applied to several test proteins (22Dauter Z. Dauter M. Rajashankar K.R. Acta Crystallogr. D. 2000; 56: 232-237Crossref PubMed Scopus (285) Google Scholar). However, hBD2 is one of the first proteins of unknown structure to be solved using this technique. The electron density map determined from experimentally derived phases was of excellent quality (Fig.1), and approximately 90% of the final structure could be automatically traced from the initial map. The remainder of the structure was built using the x-ray data collected for the native hBD2 crystal at 1.35-Å resolution. Four individual protein chains (A–D) are located within the asymmetric unit (a.u.), and all residues, except two C-terminal proline residues of the B and C chains, are traceable in the final electron density maps. The a.u. of the monoclinic form of hBD2 has a volume nearly 4 times that of the orthorhombic form. Therefore, taking into account the possible solvent contents, we assumed that hBD2 contains 14–20 monomers per a.u. Because the four monomers of the orthorhombic form exist as two topologically identical dimers within the a.u., we expected approximately eight of such dimers to be present in the a.u. of the monoclinic form. We solved phases of the monoclinic form by using the molecular replacement technique, which confirmed the presence of 16 monomers per a.u. All residues for the 16 monomers (A–P) could be easily placed in the electron density, and the structure was refined to 1.7-Å resolution. Data and refinement statistics are shown in TableI. After soaking crystals in KBr- and KI-containing solutions (immediately prior to flash freezing and data collection), bromide and iodide anions bind to similar sites on the surface of hBD2. To investigate in more detail the anion-binding sites, after calculating phases from the MAD data and refining the orthorhombic structure, we refined the coordinates of the complete model (including halide anions) against the derivative x-ray data to the maximum resolution (1.4 and 1.9 Å, respectively), using the program CNS. The anion-binding sites are located within cavities and depressions formed by mixed hydrophobic and hydrophilic environments. Coordination is mainly through van der Waals contacts, with few ionic interactions. The occupancies of the ions are relatively low (20–40% as refined by MLPHARE (Ref. 25Otwinowski Z. Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, Warrington, United Kingdom1991Google Scholar); during the CNS refinement, these occupancies were fixed to 0.5 (Ref. 29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve 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. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar)). Despite low occupancies, the high atomic numbers of the halide anions resulted in clear peaks in electron density maps calculated from the derivative x-ray data. Additionally, even though only a moderate concentration (0.25m) of KBr and KI was used in our experiments (due to problems of crystal stability in the resulting soak solution), the successful derivatization and phasing were the combined result of such factors as the overall positive charge of the protein, attracting anions from the bulk solvent, and the high resolution of anomalous data. The use of surface-bound halide anions to solve the phase problem of protein crystallography provides a simpler, relatively universal, and rapid method of solving crystal structures. Because only moderate concentrations of halides are necessary, this approach can be applied to a wide range of protein crystals. Increasing the concentration of halide anions probably would improve the strength of the anomalous signal sufficiently to allow successful structure solution using significantly lower resolution x-ray data. The hBD2 monomer displays a fold similar to that of hNP3 (17Hill C.P. Yee J. Selsted M.E. Eisenberg D. Science. 1991; 251: 1481-1485Crossref PubMed Scopus (456) Google Scholar) and bovine β-defensin-12 (bBD12) (32Zimmermann G.R. Legault P. Selsted M.E. Pardi A. Biochemistry. 1995; 34: 13663-13671Crossref PubMed Scopus (143) Google Scholar), with a three-stranded β-sheet stabilized by three disulfide bonds (Fig.2 a). We found that hBD2 contains an additional secondary-structure element, an α-helical region spanning residues Pro5–Lys10, held against the sheet by a disulfide bond formed between Cys8 and Cys37. The electron density corresponding to the majority of the structure is very well defined, and several residues could be refined in multiple conformations. Residues Arg22 and Arg23 are highly flexible, as is the entire β-turn between strands β1 and β2. The C termini are also mobile, with Lys39, Lys40, and Pro41 having elevated B factors (>40 Å2). Formation of the conserved dimer is assisted by interactions between strands β1 of both monomers, resulting in the formation of a six-stranded β-sheet. This interaction, however, only extends through two hydrogen bonds between the backbone atoms of Cys15(Fig. 2 b). We postulate that the presence of Pro17 prevents more extensive hydrogen bonding within the intermolecular sheet, thereby lowering the stability of this motif. The formation of the dimer buries 1,000–1,200 Å2, or on average 18.4% of the total surface area of the monomer, and the interface is flat and mainly hydrophobic in nature. Residues Pro5, Ala13, Ile14, Cys15, His16, and Pro17 make the majority of van der Waals contacts across the dimer interface. The monoclinic form contains two octameric assemblies within the a.u., each composed of four hBD2 dimers arranged with an approximately 422 symmetry (Fig. 3 a). The four monomers in the orthorhombic form create one half of the octameric assembly; the full octamer is generated by applying a crystallographic two-fold to the contents of the a.u. Thus, the octameric oligomer is conserved across two unique crystal forms. The octamer is formed by a mixture of hydrophobic and hydrogen bonding contacts, with residues Gly1, Asp4, Thr7, Lys10, Gly31, Leu32, Pro33, and Lys39 creating most of the contacts. The octamer is shaped like a distorted square, and is roughly 25 Å × 25 Å × 50 Å. Four inner monomers make up the majority of the octameric interfaces (1, 150–1, 800 Å2, or on average 24% of the molecular surface area of the isolated monomer), with the dimer-related outer monomers making fewer contacts (200–500 Å2, or on average 7% of the molecular surface area of the monomer). Dynamic light-scattering experiments performed on concentrated solutions of hBD2 showed a mixture of aggregated molecules, mostly populated by dimers. This result indicates that octamer formation requires additional factors. These may be, for example, the stabilizing interactions with a negatively charged membrane. The distribution of temperature factors across the octamer, lowest in the center of the octamer and increasing toward the edges, resembles the dynamic properties of many globular proteins in solution and cannot be correlated with interactions resulting from crystal formation. Additionally, a search of the Protein Data Bank for x-ray structures of proteins (except viruses) consisting of 12 or more independent molecules in the a.u. resulted in only six such structures. In all cases, multimeric molecules found in the a.u. were proven to represent biologically relevant assemblies. Therefore, it is quite likely that the hBD2 octamers common to both crystal forms correspond to stable, naturally forming oligomers of this protein. The core of the octamer is created by the N termini of the four inner monomers (chains A, C, E, G, and I, K, M, O in the monoclinic form, and chains A and C in the octameric form) (Fig. 3 a). Gly1, Gly3, Asp4 (the only acidic residue in the protein), and Thr7 create a constellation of hydrogen bonds that close the core of the octamer to solvent movement. The octamer is thus solid, and no solvent-accessible pore or channel is evident without significant movements of the protein backbone in this region. However, there are six water molecules trapped within the core of the octamer. Their mobilities are minimal, as deduced by their lowB factors, conservation in position among the three independent octamers, and coordination between protein atoms. Four of these water molecules create a cavity within the core of the octamer (Fig. 3 b). The α-helical regions of hBD2 seem to play a key role in formation of the" @default.
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• W1971891360 title "The Structure of Human β-Defensin-2 Shows Evidence of Higher Order Oligomerization" @default.
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