Structural and Functional Characterization of the Conserved Salt Bridge in Mammalian Paneth Cell α-Defensins

α-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 α-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.

␣-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 Glu 15 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.
Broad spectrum endogenous antimicrobial peptides, including defensins, contribute to the innate immune response (1). The mammalian defensins are all characterized by a central ␤-sheet that is cross-braced by an array of three disulfide bonds but can be further divided into three classes: ␣-, ␤-, and -de-fensins, based on their disulfide bond connectivities and topology (1,2). ␤-Defensins are the largest at ϳ40 amino acids and possess a Cys I -Cys V , Cys II -Cys IV , Cys III -Cys VI array (3), whereas the ␣-defensins comprise ϳ32-36 amino acids and a characteristic Cys I -Cys VI , Cys II -Cys IV , Cys III -Cys V arrangement of their disulfide bonds (4). The -defensins are considerably smaller at only 18 residues and have a Cys I -Cys VI , Cys II -Cys V , Cys III -Cys IV 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)(13)(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 posi-tions 7 and 15, respectively, in Crp4 (16 -18). The disulfide bonds maintain the ␣-defensin fold, with mutations in either the Cys I -Cys VI or Cys III -Cys V 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 Gly 19 residue is positioned in a classical ␤-bulge in the middle of ␤-strand 2. The ability of Gly to adopt a / 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).
Here, we have investigated the role of this canonical salt bridge on the native structure of ␣-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.
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 (ϳ5 ϫ 10 6 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 l 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).
NMR Spectroscopy-The samples for structure determination contained ϳ2 mg of native Crp4 or ϳ0.6 mg of (E15D)-Crp4 dissolved in either 90% H 2 O and 10% D 2 O (v/v) or 100% (v/v) D 2 O 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 pre-sented 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 2,2dimethyl-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 D 2 O. 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 pK a of Glu 15 in Crp4 and Asp 15 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 3 J HN-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 Ϫ120 Ϯ 30°for 3 J HN-H␣ greater than 8 Hz (residues Cys 4 , Tyr 5 , Cys 6 , Arg 16 , Arg 18 , Cys 21 , Leu 26 , Tyr 27 , Cys 28 , and Cys 29 for both Crp4 and (E15D)-Crp4. Additional angle restraints of Ϫ100 Ϯ 80°were included where the positive angle could be excluded based on strong sequential H␣ i-1 -HN i NOE compared with the intra residual H␣ i -HN i NOE. Side chain 1 angles and stereo-specific assignments were determined on the basis of observed NOE and 3 J H␣-H␤ coupling patterns (31). For a t 2 g 3 side chain conformation, the 1 angles were restrained to Ϫ60 Ϯ 30°(residues Cys 4 , Tyr 5 , Cys 11 , Glu 15 , Phe 25 , Cys 28 , and Cys 29 for Crp4 and residues Cys 4 , Cys 11 , Asp 15 , Arg 18 , Phe 25 , Cys 28 , and Cys 29 for (E15D)-Crp4), and for a g 2 t 3 conformation the angles were constrained to 180 Ϯ 30°(residues Val 17 , Ile 23 , and Tyr 27 for Crp4 and residues Cys 6 , Arg 13 , and Tyr 27 for (E15D)-Crp4). No residues could be confirmed to be in the g 2 g 3 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 T c 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.
Protein structures were analyzed using PROMOTIF and PROCHECK and displayed using MOLMOL. Ramachandran analysis showed that ϳ80% of the residues are in the most favored regions with the remaining in the additionally allowed (ϳ20%). 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.

NMR Spectroscopy and Resonance Assignments of Crp4 and
(E15D)-Crp4-Sets of two-dimensional NMR data were recorded for both Crp4 and (E15D)-Crp4 at 600 MHz. The spectral data were of high quality with excellent signal dispersion indicative of a well structured peptide and no additional spin systems that might indicate conformational heterogeneity (supplementary material). For both peptides full assignments of both backbone and side chain resonances were achieved using two-dimensional sequential assignment strategies. The chemical shift assignments for native Crp4 were in all cases consistent with the assignments reported by Jing et al. (23). As expected for a single point mutated peptide, the chemical shifts of most residues of (E15D)-Crp4 were very similar to those of the native peptide, but some significant differences were observed in several regions of the sequence. Most strikingly, the H⑀ side chain proton of Arg 7 , which in native Crp4 is downfield-shifted by Ͼ2 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 Arg 7 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 Arg 7 , could not be detected in the mutant. Additional significant differences in the mutant chemical shifts include the two H␤ protons of Cys 28 , which are shifted upfield by 1.5 and 1.0 ppm, H␤2 of Cys 11 (upfield 0.5 ppm), HN and both H␤s of Cys 4 (all downfield ϳ0.5 ppm), and H␣ and H␤2 of Tyr 5 (both downfield ϳ0.5 ppm). Crp4 contains one proline residue, Pro 30 , which in both the native and mutant structures was found to be in the trans-conformation as evident from strong H␣ i-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 Tyr 5 , 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 hydrogenbonded 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 (T c ) more positive than Ϫ4.6 ppb/K are involved in intramolecular hydrogen bonds (the probability increases to Ͼ93%, if Ϫ4.0 Ͻ T c Ͻ Ϫ1.0 ppb/K) (37). In Crp4 and (E15D)-Crp4 11 amides were found to have temperature coefficients Ͼ Ϫ4.6 ppb/K. The data are in good agreement with amide D 2 O exchange experiments, with seven of eight amides identified as slow exchanging, having a T c consistent with a hydrogen bond. The exception, Arg 7 , has a slow exchange with the solvent but a T c of Ϫ5.8 ppb/K. However, T c 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 Arg 7 , which at 9.88 ppm is the most downfield resonance in Crp4. For all amides identified as hydrogen bond donors by T c values or D 2 O 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  SEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38 bonded from T c data being found in turns, around the edges of the sheet, and in bulge regions.

Structures of Crp4 and (E15D)-Crp4
In addition to the temperature variation experiments, both Crp4 and (E15D)-Crp4 were subjected to pH titrations to deter-mine the pK a of Glu/Asp 15 . Favorable electrostatic interactions such as salt bridges can have a dramatic effect on the pK a values of the groups involved and by monitoring the chemical shift dependence of resonances adjacent to the titrating groups the degree of stabilization of the charged state from such interactions may be determined. Fig. 3 shows the chemical shift as a function of pH for all resonances having a significant pH dependence (Ͼ0.1 ppm) in the pH range 1.1-7.5 for Crp4 (Fig. 3A) and (E15D)-Crp4 (Fig. 3B). It is evident that the ionization states of three groups are affected by the pH changes within this range, namely His 10 , the Arg 32 C-terminal group, and Glu/Asp 15 . The data were fitted by nonlinear regression analysis, which in both Crp4 and (E15D)-Crp4 gave pK a values of ϳ5.6 and ϳ2.3 for His 10 and the C terminus, respectively. Strikingly, the Crp4 Glu 15 and (E15D)-Crp4 Asp 15 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 pK a values of Glu/Asp in Crp4 and (E15D)-Crp4 are 1.1-1.5 and 1.0 -1.7, respectively. The expected pK a values for His, ␣, ␤, and ␥ carboxyl groups are ϳ6.5, 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 Glu 15 carboxyl group is the H⑀ proton of Arg 7 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.    Given the small size of Crp4 the structure lacks a distinct hydrophobic core, and the main stabilizing features of the fold are the disulfide bonds and hydrogen bonds between the polar groups of the backbone. Some hydrophobic interactions between side chains are present, including those involving residues Cys 11 /Val 17 /Leu 26 /Cys 28 , Leu 3 /Tyr 5 /Pro 30 , and Cys 4 / Cys 6 /Arg 18 /Cys 21 /Tyr 27 . In addition, in native Crp4 an interac-tion between the Glu 15 and Arg 7 side chains can be identified. The positioning of the two side chains, which is confirmed by NOE patterns, indicates that the Glu 15 carboxylate group points toward the side of the Arg side chain, coordinating one of its oxygen atoms with the H⑀ proton and the other with one of the amino protons from the guanidinium group. The salt bridge, together with the Cys 11 -Cys 28 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 Tyr 5 side chain, which fills the void left by the larger Glu and the Arg 7 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 Tyr 5 and the positively charged Lys 12 .
The Canonical Arg 7

-Glu 15 Salt Bridge Is Not a Determinant of Crp4
Bactericidal Activity-To investigate the contribution of the Arg 7 -Glu 15 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 g/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 Arg 7 -Glu 15 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 (Arg 5 -Glu 13 ) of human ␣-defensin HNP2 have bactericidal activities equivalent to that of native HNP2 (17). Because mutagenesis of the canonical Arg 7 -Glu 15 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.
Disruption of the Arg 7 -Glu 15 Salt Bridge Does Not Induce Crp4 Susceptibility to MMP-7 Proteolysis-Mouse Paneth cell ␣-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 Arg 7 or Glu 15 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 Arg 5 /Glu 13 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
The Glu residue at position 15 is the only negatively charged residue in Crp4 and strikingly is almost completely conserved throughout the ␣-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 Glu 15 -Arg 7 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 Cys 21 and Cys 28 comprising 6 N. Zhou and H. J. Vogel, data not shown. 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 Gly 22 , which allows a classic ␤-bulge to be formed. A similar conformation is seen for Gly 19 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 ϳ1.4 Å.
Salt bridges are notoriously difficult to identify by NMR because side chain orientations are not always well defined, and proton-proton distances across salt bridges are in most cases too long to lead to detectable NOEs. However, the existence of a salt bridge may be deduced by determining the pK a of the interacting carboxylate group, which is typically lowered several pH units relative to a free carboxylate (42). By monitoring the chemical shifts as a function of pH, we determined that the pK a of Glu 15 in Crp4 is ϳ1.5, consistent with the presence of a salt bridge. The structure shows that the salt bridge interacts "side-on" with Arg 7 , which provides a definitive explanation for both the unusual shift of the Arg 7 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 Arg 7 H⑀ proton when Glu 15 is protonated support their unusually well defined position in the structure. The pH titrations also revealed unusually low pK a values for His 10 and the C-terminal carboxyl, Arg 32 . Arg 32 in Crp4 is found very close to the N terminus (Gly 1 ), and the low pK a is likely a reflection of the proximity of the positive charges of Gly 1 as well as the positive charge of its own side chain. Similarly, His 10 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 Arg 7 , Lys 12 , and Arg 13 are likely disfavoring the protonated form.
Structural Effects of the Glu 15 to Asp 15 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 Arg 7 -Glu 15 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. 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 Lys 12 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/Asp 15 carboxyl, a number of them are within the proximity and likely contribute to its very low pK a . These include Arg 7 , Lys 12 , Arg 13 , Arg 16 , Arg 18 , Arg 31 , and Arg 32 , all of which are within 8 Å of the Glu/Asp 15 carboxyl in some of the structures of the ensemble.
The observation that in HNP2 mutations disrupting the salt bridge lead to highly increased susceptibility to proteolytic degradation strongly suggests that the loop structure has been disrupted and is likely flexible. However, no such effect is seen in (E15D)-Crp4. Although structural flexibility is hard to judge from the structure itself, there is no evidence of flexibility from line broadening, lack of NOEs, or random coil-like chemical shifts in this area, strongly suggesting that the compensating interactions in (E15D)-Crp4 are enough to retain the well defined stable fold.
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 pK a of Asp 15 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.