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.     

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.     

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.     

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.     

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. (15)N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. (31)P and (2)H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.     

Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.     

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.     

Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the antimicrobial peptide gramicidin s to phospholipid bilayer membranes

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.     

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.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic beta-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK(4) to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK(4) to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK(4) to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK(4) varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK(4) can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK(4) binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Conformations of yeast alpha-mating factor and analog peptides as bound to phospholipid bilayer. Correlation of membrane-bound conformation with physiological activity

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.     

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.     

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.     

Cell selectivity correlates with membrane-specific interactions: a case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Dimer structure of magainin 2 bound to phospholipid vesicles

Magainin 2 from African clawed frog Xenopus laevis is an antimicrobial peptide with broad spectra and action mechanisms considered to permeabilize bacterial membranes. CD, vibration, and solid-state NMR spectroscopies indicate the peptide adopts an alpha-helical conformation on binding to phospholipid bilayers, and its micelle-bound conformation, being monomeric and alpha-helical, is well detailed. We showed, however, that the peptide dimerizes on binding to phospholipid bilayers. This difference in the conformation and aggregation state between micelle- and bilayer-bound states prompted us to analyze the conformation of an equipotent analog of magainin 2 (F5Y,F16W magainin 2) bound to phosphatidylcholine vesicles using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. While observed medium-range TRNOE cross peaks were characteristic of alpha-helix, many long-range cross peaks were not compatible with the peptide's monomeric state. Simulated annealing calculations generated dimer structures indicating (1) two peptide molecules have a largely helical conformation in antiparallel orientation forming a short coiled-coil structure, (2) residues 4-20 are well converged and residues 9-20 are in an alpha-helical conformation, and (3) the interface of the two peptide molecules is formed by well-defined side chains of hydrophobic residues. Finally, determined structures are compatible with numerous investigations examining magainin-phospholipid interactions.     

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.     

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.     

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.