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J. Biol. Chem., Vol. 281, Issue 38, 28068-28078, September 22, 2006
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-Defensins
124
From the
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden, the Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Australia, the ¶Departments of Pathology & Laboratory Medicine and Microbiology & Molecular Genetics, School of Medicine, University of California, Irvine, California 92697-4800, and the ||Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Received for publication, May 24, 2006 , and in revised form, July 14, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell -defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-sheet that is cross-braced by an array of three disulfide bonds but can be further divided into three classes: -, -, and 
-defensins, based on their disulfide bond connectivities and topology (1, 2). -Defensins are the largest at 
40 amino acids and possess a CysI-CysV, CysII-CysIV, CysIII-CysVI array (3), whereas the -defensins comprise 32–36 amino acids and a characteristic CysI-CysVI, CysII-CysIV, CysIII-CysV arrangement of their disulfide bonds (4). The -defensins are considerably smaller at only 18 residues and have a CysI-CysVI, CysII-CysV, CysIII-CysIV framework, combined with the remarkable feature of a head-to-tail cyclic peptide backbone, resulting from two 9-residue gene products being joined into a circle by the post-translational formation of two peptide bonds (5).
Mammalian -defensins were first identified in myeloid cells (6) but have since been found in Paneth cells of the small intestine (7, 8) and in rabbit kidney (9, 10). Paneth cell -defensins, which play an important role in enteric mucosal immunity (11), are secreted as components of granules into the lumen of small intestinal crypts in response to cholinergic stimulation or exposure to bacteria or bacterial antigens (12–14). The mouse Paneth cell -defensins, termed cryptdins (Crps),5 are secreted into the crypt lumen at concentrations of 25–100 mg/ml, orders of magnitude above their minimal inhibitory concentrations (14). Their antimicrobial activity is equivalent against Gram-positive and Gram-negative bacteria, with cryptdin-4 (Crp4) displaying the greatest mouse -defensin bactericidal activity in in vitro assays (15). The mode of action of these peptides, which involves nonspecific interactions and disruption of bacterial membranes, is dependent on peptide surface positive charge and amphipathicity, a feature common to most mammalian -defensins.
Despite the apparent positive selection of gene duplication and diversification evident in alignments of known -defensin primary structures (Fig. 1), recent studies have reported on the structural and functional roles of canonical residues conserved in all -defensins. These include the spacing and disulfide connectivities of the six Cys residues, a Gly at the position corresponding to residue 19 in Crp4, and a positively charged residue (Arg/Lys) and a negatively charged residue (Glu) found at positions 7 and 15, respectively, in Crp4 (16–18). The disulfide bonds maintain the -defensin fold, with mutations in either the CysI-CysVI or CysIII-CysV disulfide pairings, resulting in a complete disruption of the fold (16). Despite being unfolded, all disulfide-deficient Crp4 mutants retain or exceed native Crp4 bactericidal activity but are sensitive to proteolytic degradation by matrix metalloproteinase-7, the proCrp convertase (16). The conserved Gly19 residue is positioned in a classical -bulge in the middle of -strand 2. The ability of Gly to adopt a 
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angle combination not normally accessible by L-amino acids, because of its small size and less stringent conformational restrictions, is crucial for the structure of the sheet. Mutational studies on human neutrophil -defensin 2 (HNP2) have revealed that although it cannot be replaced by any other L-amino acid, a correctly folded product can be achieved by the inclusion of a D-amino acid, for which the required backbone conformation is energetically favorable (18). The final -defensin canonical feature is the occurrence of Arg and Glu, respectively, at positions 7 and 15 in mouse Crp4, which are predicted to form a conserved salt bridge (17). The role of this salt bridge in HNP2 was investigated by site-directed mutagenesis, which showed that salt bridge disruption or removal did not diminish HNP2 antibacterial activity or HNP2 precursor folding in vitro (17). However, the mutated analogues were susceptible to proteolysis by human neutrophil elastase, again emphasizing the need for a well defined stable fold to prevent attack from proteases (17).
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-defensins. To date, reported -defensin structures include the crystal structures of HNP3 (19) and HNP2 (18) and the NMR solution structures of the rabbit neutrophil defensins 2 (20) and 5 (21), the rabbit kidney defensin-1 (RK-1) (22), and mouse Crp4 (23). Although HNP2, HNP3, and RK-1 all show the presence of a salt bridge, this structural feature was not identified in Crp4 (23). After analysis of NMR data for both native Crp4 and analogues, we suggest that in the published structure a small part of the sheet has been incorrectly aligned, leading to a lack of recognition of the key salt bridge. Here we present the corrected high resolution solution structure of Crp4 and the structure of the mutant (E15D)-Crp4 in which the conserved salt bridge has been removed by effectively shortening the side chain of the Glu residue but otherwise making no change to the charge state of the native peptide. This study improves the understanding of the structural and functional roles of this conserved structural feature of the -defensin family. |
EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mutagenesis at Glu15 and Arg7 Residue Positions—Mutations were introduced into Crp4 by PCR as described previously (16) in the order described below. In the first round of mutagenesis the Crp4 construct in pET-28a (25) was used as template. In PCR 1, a mutant forward primer, e.g. C4E75D for, containing the Asp for Glu mutation at Crp4 residue position 15 flanked by three natural codons was paired with reverse primer T7 terminator (Invitrogen), a downstream sequencing primer in the pET-28a vector. In PCR 2, the mutagenizing reverse primer C4E75D rev, the reverse complement of the mutant forward primer, was paired with the T7 promoter forward primer, again from the pET-28a. Mutagenizing forward and reverse primers were: for (E15D)-Crp4, TGCAA AAGAG GAGAT CGAGT TCGTG GG (C4E75D for) and CCCAC GAACT CGATC TCCTC TTTTG CA (C4E75D rev); for (E15K)-Crp4, TGCAA AAGAG GAAAG CGAGT TCGTG GG (C4 E75K for) and CCCAC GAACT CGCTT TCCTC TTTTG CA (C4E75K rev); for (E15L)-Crp4, TGCAA AAGAG GACTA CGAGT TCGTG GG (C4E75L for) and CCCAC GAACT CGTAG TCCTC TTTTG CA (C4E75L rev); and for (E15G)-Crp4, TGCAA AAGAG GAGGA CGAGT TCGTG GG (C4E75G for) and CCCAC GAACT CGTCC TCCTC TTTTG CA (C4E75G rev). After amplification at 94 °C for 5 min, followed by successive cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 30 cycles, followed by a final extension reaction at 72 °C for 7 min, samples of purified products from reactions 1 and 2 were combined as templates in PCR 3, using T7 promoter and terminator primers as amplimers. Corresponding proCrp4 templates of these variants were prepared as described (16). All of the mutated Crp4 and proCrp4 templates were cloned in pCR-2.1 TOPO, verified by DNA sequencing, excised with SalI and EcoRI, subcloned into pET28a plasmid DNA (Novagen), and transformed into E. coli BL21(DE3)-CodonPlus-RIL cells (Stratagene) for recombinant expression (24, 25).
Purification of Recombinant Crp4 Proteins—Recombinant proteins were expressed and purified as His-tagged Crp4 fusion peptides (16, 24). Briefly, recombinant proteins were expressed at 37 °C in Terrific Broth medium by induction with 0.1 mM isopropyl -D-1-thiogalactopyranoside for 6 h at 37 °C, the cells were lysed by sonication in 6 M guanidine-HCl in 100 mM Tris-Cl (pH 8.1), and the soluble protein fraction was clarified by centrifugation (24–26). His-tagged Crp4 fusion peptides were purified using nickel-nitrilotriacetic acid (Qiagen) resin affinity chromatography (24). After CNBr cleavage, Crp4 peptides were purified by C18 reverse phase high performance liquid chromatography and quantitated by bicinchoninic acid (Pierce), and the molecular masses of purified peptides were determined using matrix-assisted laser desorption ionization mode mass spectrometry (Voyager-DE MALDI-TOF; PE Biosystems, Foster City, CA) in the Mass Spectroscopy Facility of the Department of Chemistry at the University of California, Irvine.
Bactericidal Peptide Assays—Recombinant peptides were tested for microbicidal activity against E. coli ML35 and Staphylococcus aureus 710a (27). Bacteria (5 x 106 colony-forming units/ml) resuspended in 10 mM PIPES (pH 7.4) supplemented with 0.01 volume of trypticase soy broth were incubated with test peptides in 50 µl for 1 h at 37°C, and the surviving bacteria were counted as colony-forming units/ml after overnight growth on semi-solid medium (24, 25).
Exposure of Glu15 Crp4 and proCrp4 Variants to MMP-7, the Mouse Paneth Cell Pro--defensin Convertase—Recombinant Crp4, proCrp4, and variants with site-directed mutations at position 15 were digested with MMP-7 and analyzed for evidence of proteolysis by acid-urea PAGE (24, 28). Samples of Crp4 variants (5 µg) and proCrp4 variants (11 µg) were incubated with activated recombinant human MMP-7 (0.3 1.0 µg) catalytic domain (Calbiochem, La Jolla, CA) in buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2 for 18–24 h at 37 °C, and equimolar samples of all digests were analyzed by acid-urea PAGE (16, 24).
NMR Spectroscopy—The samples for structure determination contained 2 mg of native Crp4 or 0.6 mg of (E15D)-Crp4 dissolved in either 90% H2O and 10% D2O (v/v) or 100% (v/v) D2O at pH 4.5. All of the spectral data were recorded at 600 and 500 MHz on Bruker Avance NMR spectrometers. Two-dimensional experiments recorded included double quantum filtered correlation spectroscopy (DQF-COSY), TOCSY using an MLEV-17 spin lock sequence with a mixing time of 80 ms, and NOESY with mixing times of 100, 150, or 200 ms. Selected spectral data, including preliminary data on an additional salt bridge-deficient analogue, (R7G)-Crp4, are presented as supplementary material. The spectra were generally acquired with 4096 complex data points in F2 and 512 increments in the F1 dimension over a spectral width of 12 ppm. The spectra were processed on a Silicon Graphics Octane work station using XWINNMR (Bruker). The F1 dimension was generally zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied before Fourier transformation. Chemical shifts were internally referenced to 2,2-dimethyl-2-silapentane sulfonic acid (DSS) at 0.00 ppm. Slowly exchanging NH protons were detected by acquiring a series of one-dimensional and TOCSY spectra of the fully protonated peptide immediately after dissolution in D2O. Further evidence for hydrogen bonds was deduced from amide temperature coefficients, which were determined by recording TOCSY spectra at 288, 293, 298, 303, and 308 K and plotting the amide shifts as a function of temperature. The pKa of Glu15 in Crp4 and Asp15 in (E15D)-Crp4 and the C-terminal group were estimated by monitoring the effects of pH in the range of 1.1–7.5 on the chemical shifts of resonances within the vicinity of the carboxylate group.
Structure Determination—Distance restraints for Crp4 were derived primarily from 150-ms NOESY spectra recorded at 298 K and 600 MHz with additional restraints derived from a NOESY recorded at 500 MHz with a cryogenic probe added during refinement stage. Distance restraints for (E15D)-Crp4 were derived from a 600-MHz NOESY recorded at 298 K with a mixing time of 200 ms to compensate for the significantly lower sample concentration. The spectral data were analyzed, and the cross-peaks were assigned and integrated in CARA (29) and converted to distance restraints using DYANA (30). Backbone dihedral restraints were inferred from 3JHN-H coupling constants derived from either the one-dimensional or a high digital resolution double quantum filtered correlation spectroscopy. The dihedral angle was restrained to –120 ± 30° for 3JHN-H greater than 8 Hz (residues Cys4, Tyr5, Cys6, Arg16, Arg18, Cys21, Leu26, Tyr27, Cys28, and Cys29 for both Crp4 and (E15D)-Crp4. Additional angle restraints of –100 ± 80° were included where the positive angle could be excluded based on strong sequential Hi-1-HNi NOE compared with the intra residual Hi-HNi NOE. Side chain 
1 angles and stereo-specific assignments were determined on the basis of observed NOE and 3JH-H coupling patterns (31). For a t2g3 side chain conformation, the 1 angles were restrained to –60 ± 30° (residues Cys4, Tyr5, Cys11, Glu15, Phe25, Cys28, and Cys29 for Crp4 and residues Cys4, Cys11, Asp15, Arg18, Phe25, Cys28, and Cys29 for (E15D)-Crp4), and for a g2t3 conformation the angles were constrained to 180 ± 30° (residues Val17, Ile23, and Tyr27 for Crp4 and residues Cys6, Arg13, and Tyr27 for (E15D)-Crp4). No residues could be confirmed to be in the g2g3 conformation based on experimental data. Hydrogen bonds were included into the structure calculations for all of the amide protons concluded to be slow exchanging or having a Tc consistent with a hydrogen bond, only once a suitable acceptor could be identified in the preliminary structures. In all cases these hydrogen bonds were found between the backbone atoms within the elements of secondary structure.
Three-dimensional structures were calculated using simulated annealing and energy minimization protocols from ARIA (32) within the program CNS (33) as described previously (34). The protocol involved a high temperature phase comprising 4000 steps of 0.015 ps of torsion angle dynamics, a cooling phase with 4000 steps of 0.015 ps of torsion angle dynamics during which the temperature is lowered to 0 K, and finally an energy minimization phase comprising 5000 steps of Powell minimization. The refinement in explicit water involves the following steps: first, heating to 500 K via steps of 100 K, each comprising 50 steps of 0.005 ps of Cartesian dynamics; second, 2500 steps of 0.005 ps of Cartesian dynamics at 500 K, before a cooling phase where the temperature is lowered in steps of 100 K, each comprising 2500 steps of 0.005 ps of Cartesian dynamics; finally, the structures were minimized with 2000 steps of Powell minimization.
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80% of the residues are in the most favored regions with the remaining in the additionally allowed (20%). The coordinates representing the solution structure of Crp4 and (E15D)-Crp4 and the experimental restraints have been submitted to the Protein Data Bank and given the access codes 2GW9 and 2GWP, respectively. |
RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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side chain proton of Arg7, which in native Crp4 is downfield-shifted by >2 ppm (9.61 ppm), is found at 7.24 ppm in the mutant, close to the expected random coil shift of Arg residues. Other signals from the Arg7 side chain are shifted downfield in the mutant by between 0.7 and 0.2 ppm, and a sharp resonance at 6.55 ppm, which originated from the guanidinium group of Arg7, could not be detected in the mutant. Additional significant differences in the mutant chemical shifts include the two H protons of Cys28, which are shifted upfield by 1.5 and 1.0 ppm, H2 of Cys11 (upfield 0.5 ppm), HN and both Hs of Cys4 (all downfield 0.5 ppm), and H and H2 of Tyr5 (both downfield 0.5 ppm). Crp4 contains one proline residue, Pro30, which in both the native and mutant structures was found to be in the trans-conformation as evident from strong Hi-1-H
i NOEs to the preceding residue.
The presence of secondary structure in peptides can generally be readily identified by an analysis of the deviation of the H shifts from random coil shifts (35). Fig. 2 shows the secondary H shifts for Crp4 and (E15D)-Crp4, from which it is clear that the general trend is stretches of positive values, consistent with the triple-stranded -sheet that is typical of an -defensin fold. With the exception of Tyr5, only small differences in the H secondary shifts are seen between the two peptides, suggesting that despite the large effects on the chemical shifts of some residues, the mutation does not significantly affect the backbone fold.
Temperature Variation and pH Titration Studies of Crp4 and (E15D)-Crp4—Monitoring the amide chemical shifts of a protein as a function of temperature is a rapid and powerful method for identifying hydrogen bond donors in a three-dimensional structure, because intramolecularly hydrogen-bonded amides have a low sensitivity to temperature (36, 37). For both Crp4 and (E15D)-Crp4 the temperature dependences of the amide chemical shifts were determined from TOCSY spectra over the temperature range 288–308 K. Generally 85% of amides that have a temperature coefficient (Tc) more positive than –4.6 ppb/K are involved in intramolecular hydrogen bonds (the probability increases to >93%, if –4.0 < Tc <–1.0 ppb/K) (37). In Crp4 and (E15D)-Crp4 11 amides were found to have temperature coefficients >–4.6 ppb/K. The data are in good agreement with amide D2O exchange experiments, with seven of eight amides identified as slow exchanging, having a Tc consistent with a hydrogen bond. The exception, Arg7, has a slow exchange with the solvent but a Tc of –5.8 ppb/K. However, Tc values are known to be affected by strong shielding/deshielding (36) and may give false positives/negatives if the amide resonance has an unusual shift. This is the case for Arg7, which at 9.88 ppm is the most downfield resonance in Crp4. For all amides identified as hydrogen bond donors by Tc values or D2O exchange, suitable acceptors were identified within elements of secondary structure in the preliminary structures, with the slow exchanging amides being part of the core of the -sheet and the additional amides identified as hydrogen bonded from Tc data being found in turns, around the edges of the sheet, and in bulge regions.
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5.6 and 2.3 for His10 and the C terminus, respectively. Strikingly, the Crp4 Glu15 and (E15D)-Crp4 Asp15 carboxyl groups are largely unaffected as the pH is lowered to 3, and as a result the full titration curves cannot be obtained without subjecting the proteins to extreme conditions. However, based on curve fitting of the available data for several resonances affected by protonation/deprotonation, the 95% confidence intervals for the pKa values of Glu/Asp in Crp4 and (E15D)-Crp4 are 1.1–1.5 and 1.0–1.7, respectively. The expected pKa values for His, , , and 
carboxyl groups are 6.5, 3.5–4.3, 3.9–4.0, and 4.3–4.5, respectively (38, 39); hence the noncharged state of the His and the charged state of both the termini and the Glu/Asp are significantly stabilized by interactions in the folded structure. The resonance that is most affected by the titration of the Glu15 carboxyl group is the H proton of Arg7 in native Crp4, which at low pH start to move back from its downfield shifted position toward its random coil value.
Structure Determination and Description of the Three-dimensional Structure—From the NMR data a set of restraints including upper limit distance restraints based on NOE cross-peak intensities, backbone and side chain 1 dihedral angles, and hydrogen bond restraints was derived and used for structure determination of the two peptides. Both structures were calculated by simulated annealing and refined in explicit solvent and the structural and energetic statistics for the final families of 20 structures are summarized in Table 1. All of the structures are in good agreement with the experimental data and have good covalent geometries, as evident from low deviations from optimal bond lengths and angles and from the Ramachandran statistics.
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-sheet. The sheet is made up by strands comprising residues 5–8, 15–21, and 25–29, with the second strand having two bulges around residues 18–19 and 21–22. The conformation of these bulges requires Gly residues at positions 19 and 21, because of the ability of Gly to adopt backbone conformations normally not accessible by other L-amino acids. The three strands are linked by two well defined -turns comprising residues 12–15 and 22–25, both of which adopt a type II conformation, and one -turn comprising residues 8–10. The arrangement of the sheet and turns is supported by a large number of cross-strand NOEs, as illustrated in Fig. 5. Also indicated in Fig. 5 is the hydrogen bond network that was identified by analysis of amide exchange, amide proton Tc values, and structure calculations.
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proton and the other with one of the amino protons from the guanidinium group. The salt bridge, together with the Cys11-Cys28 disulfide bond, appears to stabilize the residue 9–15 loop, which is the only part of the molecule, apart from the two termini, not involved in elements of regular secondary structure. In (E15D)-Crp4 the shortened carboxyl-bearing side chain makes this interaction impossible, and the mutation results in a reorientation of the Tyr5 side chain, which fills the void left by the larger Glu and the Arg7 side chains, with the latter moving away from the molecular core, out into solution. Electrostatic interactions that apparently compensate for the lack of the salt bridge are formed between the Asp and the phenolic hydrogen of Tyr5 and the positively charged Lys12.
The Canonical Arg7–Glu15 Salt Bridge Is Not a Determinant of Crp4 Bactericidal Activity—To investigate the contribution of the Arg7–Glu15 salt bridge to Crp4 microbicidal function, the bactericidal activities of native Crp4 and Crp4 variants with salt bridge disruptions were compared with E. coli and S. aureus in in vitro assays (Fig. 6). Under the conditions of these assays, the overall bactericidal activities of Crp4, (E15D)-Crp4, (E15L)-Crp4, and (E15G)-Crp4 were similar, reducing bacterial cell survival by at least three log values at or below 10 µg/ml (Fig. 6 and data not shown). The results of additional assays performed against strains of Vibrio cholerae, Listeria monocytogenes, and wild-type Salmonella enterica serovar Typhimurium were reproducibly similar to those in Fig. 6, as were assays performed with a (R7G)-Crp4 variant, which also contains a salt bridge disruption (data not shown). These findings show that Crp4 bactericidal activity is independent of the Arg7–Glu15 salt bridge, although the dose-response curves of certain peptides differed modestly (Fig. 6). This finding is consistent with the fact that corresponding salt bridge mutants (Arg5-Glu13) of human -defensin HNP2 have bactericidal activities equivalent to that of native HNP2 (17). Because mutagenesis of the canonical Arg7–Glu15 salt bridge had little or no effect on Crp4 bactericidal action, alternative roles for the salt bridge were considered, including protection from proteolysis by matrix metalloproteinase-7 (MMP-7), the activating convertase for mouse Paneth cell pro--defensins.
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-defensin biosynthesis requires MMP-7-mediated proteolytic conversion of inactive proCrps to their functionally active forms (40, 41). Because mutations to the mouse Crp4 disulfide array result in Crp4 proteolysis by MMP-7 (16) and because mutations in the HNP2 salt bridge induce susceptibility to neutrophil elastase, the hypothesis that Arg7 or Glu15 mutants of Crp4 and proCrp4 would be subject to MMP-7-mediated degradation was tested. Native Crp4 is completely resistant to MMP-7 in vitro, and MMP-7 activates native proCrp4 without cleaving within the -defensin moiety of the precursor (Fig. 7 and Refs. 16, 24, 25, and 41). In contrast to the sensitivity of Arg5/Glu13 HNP2 variants (17), none of the Crp4 or proCrp4 salt bridge variants tested, including (E15D)-Crp4, (R7G)-Crp4, (E15G)-Crp4, (E15D)-proCrp4, (R7A)-proCrp4, and (R7G)-proCrp4, displayed evidence of proteolysis by MMP-7 in this highly sensitive assay (Fig. 7). These findings suggest that structural alterations induced in Crp4 by disrupting the canonical -defensin salt bridge do not ensure sensitivity to proteolysis per se, but that variations in peptide primary structure exclusive of canonical positions, in particular the strong electropositive charge of Crp4, also contribute to the proteolytic stability. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-defensin family. Based on structural data, this Glu has been proposed to be involved in a conserved salt bridge with an Arg/Lys, and in a recent study a number of mutants of HNP2 exploring the role of this salt bridge were generated and analyzed with respect to bactericidal activity, in vitro folding ability, and proteolytic stability (17). Here, we have investigated the biological and structural consequences of removing this conserved salt bridge in a mouse Paneth cell -defensin, Crp4. The NMR structure of native Crp4 was recently reported, but apparent misassignment of a few crucial hydrogen bonds between the 2 and 3 strands resulted in a small part of one strand being incorrectly aligned with the sheet, and as a consequence, the Glu15-Arg7 salt bridge was not identified (23). However, as we show here, the conserved salt bridge is indeed present in the corrected structure of Crp4, and its structural role has been evaluated by structural analysis of the analogue (E15D)-Crp4, in which it has been removed.
The Structure of Crp4 and Comparison with Other -Defensin Structures—The misassignment of a few hydrogen-bonding partners in the earlier reported Crp4 solution structure by Jing et al. (23) led to a different arrangement of a small part of the 2 strand with respect to the 3 strand. Nevertheless the overall shape of the molecule was very similar to that obtained in the present study. Unexpectedly the incorrect alignment of these two strands did not give rise to obvious problems in the energy of the original structure. Because the hydrogen bonds were included in all the final structure calculations the other restraints used (e.g. NOEs) did not move the structure toward a more correct positioning of the sheet. However, in subsequent unrestrained molecular dynamics calculations, movement of the 2 strand and increased hydrogen bonding in Crp4 was observed. Movement of strands is not normally observed in such unrestrained molecular dynamics simulations, indicating that part of the original structure had considerable strain associated with it. During this movement process the other two strands of the sheet remained in their correct orientation, which is identical to that observed in this study. After 6 ns the relaxed structure resembled the one reported here and this remained stable during a further 14 ns of simulation time.6 The results obtained in the unrestrained simulations are clearly consistent with the outcome of the current structural study of Crp4.
The present solution structure of Crp4 adopts a typical -defensin fold that is characterized by a triple-stranded antiparallel -sheet. Fig. 8 shows a comparison of Crp4, HNP3, and RK-1, and it is clear that the most significant difference between Crp4 and most other -defensin is in the hairpin region. This is a direct result of the loop between Cys21 and Cys28 comprising only six residues in Crp4 compared with nine in HNP3 and eight in RK-1. The longer loop changes not only the structure of the hairpin turn but also quite dramatically the direction in which the turn projects away from the core of the molecule. Although the number of residues in this loop in HNP3 allows the two anti-parallel strands to be linked by a regular -turn, the lack of three residues, an odd number, means that in Crp4 a bulge has to be formed for the turn to be able to adopt a regular conformation. This is facilitated by Gly22, which allows a classic -bulge to be formed. A similar conformation is seen for Gly19 in all known -defensin structures, and it has been shown to be crucial for the structure of the sheet and thus is likely responsible for the evolutionary conservation of a Gly at this position (18). The structure of the molecular core with the central -sheet and the disulfide bonds is highly conserved, and the three molecules superimpose over this region with a root mean square deviation of 1.4 Å.
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1.5, consistent with the presence of a salt bridge. The structure shows that the salt bridge interacts "side-on" with Arg7, which provides a definitive explanation for both the unusual shift of the Arg7 H proton and the slow exchange behavior observed for the amino protons from the guanidinium group. Both protons have several NOEs to surrounding groups, and the large chemical shift changes observed for the Arg7 H proton when Glu15 is protonated support their unusually well defined position in the structure. The pH titrations also revealed unusually low pKa values for His10 and the C-terminal carboxyl, Arg32. Arg32 in Crp4 is found very close to the N terminus (Gly1), and the low pKa is likely a reflection of the proximity of the positive charges of Gly1 as well as the positive charge of its own side chain. Similarly, His10 does not have a direct interaction with another charged group, but the overall positive nature of Crp4 and the presence of several close by positive charges including Arg7, Lys12, and Arg13 are likely disfavoring the protonated form.
Structural Effects of the Glu15 to Asp15 Mutation in Crp4—With the only exception of two sequences from guinea pig and one from rhesus enteric defensin-6, the Arg/Lys-Glu pair of oppositely charged residues is conserved throughout more than 40 known -defensins. Interestingly, no members of the family have an Asp rather than a Glu at the corresponding position, despite the two residues only differing in a single base pair on a genetic level and both potentially having the ability to form salt bridges with positively charged residues. This observation leads to the question: what is the role of the salt bridge in the mammalian -defensins? In a recent study on HNP2, several mutants disrupting the salt bridge were generated, and it was found that the salt bridge is not needed for biological activity nor in vitro folding of HNP2 precursors, although the in vitro folding of mature domains was affected (17). In contrast, the mutated analogues were more susceptible to degradation by neutrophil elastase, a major protease that colocalizes with HNP2 in neutrophil azurophilic granules, suggesting that the salt bridge may contribute to protease resistance in vivo. Here, despite clear structural implications of a conservative substitution at the Arg7–Glu15 salt bridge disruption to the Crp4 9–15 loop, biological and biochemical consequences of those structural modifications were not evident from assays of bactericidal activity and in vitro proteolytic precursor activation of Crp4 salt bridge variants, strongly suggesting a different primary role for the salt bridge at least in the case of the more highly cationic mouse Paneth cell -defensins.
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-sheet structures. However, although the secondary shifts (Fig. 2) are generally very similar in the two peptides, there are some significant differences, both locally around the mutations and in other regions, confirming structural changes at a minimum associated with side chain orientations. Fig. 9 shows a comparison of the lowest energy structures of native Crp4 and (E15D)-Crp4 and illustrates the main differences around the mutation. Although the projection of the Asp15 side chain does not differ between the two peptides, Arg7 has rotated, and its charged side chain projects into the solution in the mutant structure. However, the most striking difference is the position of the Tyr5 side chain, which folds in and fills the space left by the mutation. As a result a large number of new NOEs are seen for the Tyr side chain aromatic protons, and its position explains the large chemical shift differences observed in (E15D)-Crp4. Tyr5 forms new interactions with the Cys11–Cys28 disulfide, as well as Arg7 and Pro30 side chains. The observation that the Asp15 pKa in (E15D)-Crp4 is similar to what is seen for Glu15 in native Crp4 was initially a surprise. However, from the structure it is clear that new interactions that stabilize the charged state of Asp15 are formed. The reorientation of Tyr5 allows a strong electrostatic interaction between the Asp15 and the phenolic hydrogen on the Tyr5 side chain. Interactions between Asp and hydrogen bond donors can be highly stabilizing in protein structures (42). Furthermore, the positively charged Lys12 side chain can interact closely with the Asp/Glu15 carboxyl. Although Lys12 is in the proximity of the Glu/Asp15 in both Crp4 and (E15D)-Crp4, it appears from the structure that they interact more closely in (E15D)-Crp4. This observation is supported by the fact that in (E15D)-Crp4 the Lys12 side chain displays a chemical shift separation of both the and methylene protons, strongly suggesting an ordered conformation. This is in contrast to what is seen in Crp4 where the Lys12 side chain appears to be moving freely in solution, and as a result the geminal methylene protons have identical chemical shifts. A hydrogen bond between the Lys12 backbone amide and the Glu/Asp carboxyl group is present in both peptides. In addition it should be noted that both Crp4 and (E15D)-Crp4 have a large number of positive charges, and although they are not in direct contact with the Glu/Asp15 carboxyl, a number of them are within the proximity and likely contribute to its very low pKa. These include Arg7, Lys12, Arg13, Arg16, Arg18, Arg31, and Arg32, all of which are within 8 Å of the Glu/Asp15 carboxyl in some of the structures of the ensemble.
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Concluding Remarks—In summary we have reported the solution structures and biochemical properties of Crp4 and a salt bridge-deficient (E15D)-Crp4 mutant. Despite the extensive conservation of this salt bridge throughout the -defensin peptide family, our findings show that its disruption does not significantly affect the overall fold of the peptide but does induce some local structural changes involving side chains around the point of the mutation. These changes compensate for the lack of the salt bridge and appear to stabilize the fold as evident from the apparent well defined structure, as well as the low pKa of Asp15 in (E15D)-Crp4. The NMR data are consistent with a stable structure and lacks evidence of dynamic processes such as additional resonances from multiple conformations and exchange broadening. Furthermore, mutations of the salt bridge do not affect in vitro biological activity or the proteolytic stability of Crp4, which has been shown to be the case in HNP2 (17). Although HNP2 precursors with a disrupted salt bridge can be folded in vitro (17), perhaps the conserved salt bridge is under positive selection in -defensins to facilitate folding or trafficking in the endoplasmic reticulum in vivo rather than determining the stability or activity of the final folded product.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains five supplementary figures.
The atomic coordinates and structure factors (codes 2GW9 and 2GWP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
2 National Health and Medical Research Council, Australia industry fellow.
3 Recipient of a Scientist Award from the Alberta Heritage Foundation for Medical Research.
4 An Australian Research Council Professorial Fellow.
1 To whom correspondence should be addressed. Tel.: 46-480-446152; Fax: 46-480-446262; E-mail: johan.rosengren{at}hik.se.
5 The abbreviations used are: Crp, cryptdin; HNP, human neutrophil -defensin; RK, rabbit kidney defensin; PIPES, 1,4-piperazinediethanesulfonic acid; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; MMP, matrix metalloproteinase; TOCSY, total correlation spectroscopy.
6 N. Zhou and H. J. Vogel, data not shown.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, Crp4; •, (E15D)-Crp4; 
, (E15L)-Crp4; 
, (E15G)-Crp4.
