Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins: solution structures of mouse CRYPTDIN-4 and (E15D)-CRYPTDIN-4

alpha-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 alpha-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.     

Solution structure of cryptdin-4, a mouse paneth cell alpha-defensin

Mammalian defensins are abundant antimicrobial peptides that contribute to host defense. They are characterized by several conserved amino acids, including six invariant cysteine residues which form three intramolecular disulfide bonds and stabilize the tertiary structure. Cryptdin-4 (Crp4), a mouse alpha-defensin with potent in vitro bactericidal activity, has a primary structure distinct from all known alpha-defensins in that its polypeptide backbone uniquely lacks three residues between Cys(IV) and Cys(V). NMR diffusion experiments showed that Crp4 is monomeric in solution, and its three-dimensional solution structure, determined by two-dimensional proton NMR, consists of a triple-stranded antiparallel beta-sheet with the beta-strands joined to each other by a series of tight turns and a beta-hairpin. However, the overall beta-sheet content in Crp4 is lower than that of other alpha-defensin structures, while the shape and orientation of the Crp4 beta-hairpin also differ from those of other alpha-defensin structures. These structural characteristics combined with the high overall cationicity of Crp4 may contribute to its broad bactericidal spectrum and membrane disruptive activity.     

Matrix metalloproteinase-7 activation of mouse paneth cell pro-alpha-defensins: SER43 down arrow ILE44 proteolysis enables membrane-disruptive activity

Small intestinal Paneth cells secrete alpha-defensin microbicidal peptides as mediators of innate enteric immunity. In mice, production of mature Paneth cell alpha-defensins, termed cryptdins (Crps), requires proteolytic activation of inactive precursors (pro-Crps) by the convertase matrix metalloproteinase-7. Proteolysis of mouse (pro-Crp4)(20-92) produces the specific cleavage intermediates pro-Crp4(44-92), pro-Crp4(54-92), and pro-Crp4(59-92). To identify which cleavage event enables bactericidal activity, recombinant pro-Crp4-processing intermediates were purified to homogeneity and assayed for bactericidal peptide activity. The in vitro bactericidal activities of pro-Crp4-processing intermediates were very similar to fully processed Crp4, contrasting the lack of bactericidal and membrane-disruptive activity shown by pro-Crp4(20-92). Thus, cleavage of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) is sufficient to activate bactericidal activity, and amino acids in the pro-Crp4(20-43) of the proregion maintain the precursor in an inactive state. Because cationic Arg residues are determinants of Crp4 bactericidal peptide activity, we hypothesized that Asp and Glu residues in pro-Crp4(20-43) neutralize Crp4 Arg side chains in pro-Crp4(20-92). Therefore, a pro-Crp4(20-92) variant with Gly substitutions at all pro-Crp4(20-43) Asp and Glu positions ((DE/G)-pro-Crp4) was prepared, and it was bactericidal and lysed phospholipid vesicles under conditions where native pro-Crp4(20-92) lacks activity. These findings show that MMP-7 proteolysis of pro-Crp4(20-92) at Ser(43) downward arrowIle(44) converts inactive precursors to bactericidal forms by removal of covalently associated, inhibitory acidic amino acids from proximity with the Crp4 component of the molecule.     

Quantitative interactions between cryptdin-4 amino terminal variants and membranes

Paneth cells secrete alpha-defensins into the lumen from the base of small intestinal crypts, and cryptdin-4 (Crp4) is the most potent mouse alpha-defensin in vitro. Purified recombinant Crp4 and Crp4 variants with (des-Gly)-, (Gly1Val)-, (Gly1Asp)-, and (Gly1Arg)-substitutions were all bactericidal with Crp4 and (Gly1Arg)-Crp4 being slightly more active than other variants. Bactericidal activities correlated directly with permeabilization of live Escherichia coli, with equilibrium binding to E. coli membrane phospholipid bilayers and vesicles, and with induced graded fluorophore leakage from phospholipid vesicles. The Crp4 peptide N-terminus affects bactericidal activity modestly, apparently by influencing peptide binding to phospholipid bilayers and subsequent permeabilization of target cell membranes.     

Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes

The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC, MMP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.     

Structure-activity determinants in paneth cell alpha-defensins: loss-of-function in mouse cryptdin-4 by charge-reversal at arginine residue positions

Paneth cells secrete microbicidal enteric alpha-defensins into the small intestinal lumen, and cryptdin-4 (Crp4) is the most bactericidal of the mouse alpha-defensin peptides in vitro. Here, site-directed Arg to Asp mutations in Crp4 have been shown to attenuate or eliminate microbicidal activity against all of the bacterial species tested regardless of the Arg residue position. R31D/R32D charge-reversal mutagenesis at the C terminus and mutations at R16D/R18D, R16D/R24D, and R18D/R24D in the Crp4 polypeptide chain eliminated in vitro bactericidal activity, blocked peptide-membrane interactions, as well as Crp4-mediated membrane vesicle disruption. Lys for Arg charge-neutral substitutions in (R16K/R18K)-Crp4 did not alter the bactericidal activity relative to Crp4, showing that bactericidal activity appears not to require the guanidinium side chain of Arg at those two positions. Partial restoration of (R31D/R32D)-Crp4 bactericidal activity occurred when an electropositive Arg for Gly substitution was introduced at the peptide N terminus and the (G1R/R31D/R32D)-Crp4 peptide exhibited intermediate membrane binding capability. Also, the loss of peptide bactericidal activity in (G1D/R31D/R32D)-Crp4 and (R16D/R24D)-Crp4 mutants corresponded with diminished phospholipid vesicle disruptive activity. Fluorophore leakage from anionic phospholipid vesicles induced by the charge-reversal variants was negligible relative to Crp4 and lower than that induced by pro-Crp4, the inactive Crp4 precursor. Thus, Arg residues function as determinants of Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. The role of the Arg residues, however, was surprisingly independent of their position in the polypeptide chain.     

Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.     

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 ?) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.     

In vitro activation of the rhesus macaque myeloid alpha-defensin precursor proRMAD-4 by neutrophil serine proteinases

Alpha-defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-alpha-defensin-4 (proRMAD-4((20-94))) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4((20-94)) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4((62-94)). The final CG cleavage product was RMAD-4((55-94)), and P3 produced both RMAD-4((55-94)) and RMAD-4(57-94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4((62-94)), RMAD-4((55-94)), and RMAD-4(57-94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4((20-94)) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4((20-94)) and (DE/A)-proRMAD-4((20-94)) were as active as mature RMAD-4((62-94)). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4((20-94)), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4((62-94)) resists NE, CG, and P3 proteolysis completely, RMAD-4((62-94)) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the alpha-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-alpha-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.     

Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7

The bactericidal activity of mouse Paneth cell alphadefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH(2)-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitro bactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser(43) downward arrow Ile(44) and Ala(53) downward arrow Leu(54) in the proregion and near the Crp4 peptide NH(2) terminus between Ser(58) downward arrow Leu(59). Because the Crp4 NH(2) terminus occurs at Gly(61), not Leu(59), MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58 downward arrow L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser(43) downward arrow Val(44) and Ser(53) downward arrow Leu(54) sites but not at the peptide NH(2) terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu(54) at its NH(2) terminus resulting from Ala(53) downward arrow Leu(54) cleavage and loss-of-function at the Ser(58) downward arrow Ser(59) cleavage site. Thus, alpha-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser(43) downward arrow Val(44) completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser(43) downward arrow Val(44). These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor.     

The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein–protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg⁷ or Glu¹⁵ residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg⁷ and Glu¹⁵ substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.     

Arginine in α-defensins: differential effects on bactericidal activity correspond to geometry of membrane curvature generation and peptide-lipid phase behavior

The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded β-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In mouse cryptdin-4 (Crp4) and rhesus myeloid α-defensin-4 (RMAD4), complete substitutions of Arg with Lys affect bactericidal peptide activity very differently. Lys-for-Arg mutagenesis attenuates Crp4, but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys-for-Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, small-angle x-ray scattering analyses showed that Arg-to-Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared with the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by Lys-for-Arg mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between small-angle x-ray scattering measurements and biological activity provides evidence that specific types of α-defensin-induced membrane curvature-generating tendencies correspond directly to bactericidal activity via membrane destabilization.     

The alternative sigma factor sigma is required for resistance of Salmonella enterica serovar Typhimurium to anti-microbial peptides

The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters a variety of anti-microbial peptides during the course of infection. We report here that the extracytoplasmic sigma factor sigma(E) (RpoE) is required for Salmonella resistance to killing by the bactericidal/permeability-increasing protein (BPI)-derived peptide P2 and the murine alpha-defensin cryptdin-4 (Crp4). Moreover, sigma(E)-deficient S. Typhimurium is attenuated for virulence after oral infection of immunocompromised gp91phox(-/-) mice that lack a functional NADPH phagocyte oxidase, suggesting that sigma(E) plays an important role in resistance to non-oxidative mucosal host defences such as anti-microbial peptides. Although both P2 and Crp4 target the cell envelope, bacterial killing by these peptides appears to occur by distinct mechanisms. Formate enhances bacterial resistance to P2, as previously demonstrated, but not to Crp4. Both sigma(E) and cytoplasmic membrane-associated formate dehydrogenase are required for the protective effect of formate against P2. In contrast to P2, Crp4 does not inhibit bacterial respiration at lethal concentrations. However, both peptides induce expression of rpoE, suggesting that they trigger a common mechanism for sensing extracytoplasmic stress.     

Kinetics of cryptdin-4 translocation coupled with peptide-induced vesicle leakage

The antimicrobial peptide cryptdin-4 (Crp4), a member of the alpha-defensin family, is shown to translocate cooperatively across phospholipid bilayers. The cooperativity of the process is manifested by translocation kinetics which vary with the peptide to lipid molar ratio. A simple association model suggests dimerization. Black lipid membrane experiments reveal that Crp4 translocation does not create well-defined aqueous pores, as is often common among peptides exhibiting cooperative translocation. Still, the efflux induced by Crp4 upon its interaction with fluorophore-loaded vesicles is shown to be a direct result of the membrane perturbation resulting from the translocation process. Leakage can be predicted by relating membrane permeability to the fraction of peptide translocated. Crp4 translocation has implications for its antimicrobial activity as internalized peptide would be available to attack intracellular targets.     

Activation of Paneth cell alpha-defensins in mouse small intestine.

Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.     

Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed

A 7Crp peptide composed of seven major human T cell epitopes derived from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 is an ideal tolerogen for peptide immunotherapy against Japanese cedar pollinosis. To maximize the accumulation level of the 7Crp peptide in transgenic rice seed, we tested endosperm specific promoters and intracellular localizations suitable for stable accumulation. A 7Crp peptide carrying the KDEL ER retention signal directed by the 2.3-kb promoter of the glutelin GluB-1, which contains a signal peptide, accumulated at the highest level of about 60 μg/grain. Notably, the 7Crp peptide predominantly accumulated in ER-derived protein bodies irrespective of the presence of various sorting signals or expression as a fusion protein with glutelin. We attribute this abnormal pattern of accumulation to the formation of disulfide bonds between the 7Crp peptide and cysteine-rich (Cys-rich) prolamin storage proteins. Furthermore, the formation of these aggregates induced the chaperone proteins BiP and PDI as an ER stress response.     

Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial alpha-defensin

Defensins represent an evolutionarily conserved group of small peptides with potent antibacterial activities. We report here that extracellular proteinases secreted by the human pathogens Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus pyogenes release dermatan sulphate by degrading dermatan sulphate-containing proteoglycans, such as decorin. Dermatan sulphate was found to bind to neutrophil-derived alpha-defensin, and this binding completely neutralized its bactericidal activity. During infection, proteoglycan degradation and release of dermatan sulphate may therefore represent a previously unknown virulence mechanism, which could serve as a target for novel antibacterial strategies.     

The conserved salt bridge in human alpha-defensin 5 is required for its precursor processing and proteolytic stability

Mammalian alpha-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical beta-bulge are totally conserved in the otherwise diverse sequences of all known mammalian alpha-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg(6)-Glu(14) salt bridge in human alpha-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg(13)-Gln(14) peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature alpha-defensin in vivo.     

Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1)

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.