Bryan Ericksen

Antibacterial Activity and Specificity of the Six Human α-Defensins

ABSTRACT We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human α-defensins concurrently on a single microplate. The defensins were prepared by solid-phase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD50s), LD90s, LD99s, and LD99.9s obtained from colony counts. On the basis of their respective vLD90s and vLD99s, the relative potencies of human myeloid α-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 = HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five α-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human α-defensins and raised intriguing questions about the in vivo function(s) of HD6.

Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins

Despite the small size and conserved tertiary structure of defensins, little is known at a molecular level about the basis of their functional versatility. For insight into the mechanism(s) of defensin function, we prepared enantiomeric pairs of four human defensins, HNP1, HNP4, HD5, and HBD2, and studied their killing of bacteria, inhibition of anthrax lethal factor, and binding to HIV-1 gp120. Unstructured HNP1, HD5, and HBD3 and several other human α- and β-defensins were also examined. Crystallographic analysis showed a plane of symmetry that related LHNP1 and DHNP1 to each other. Either d-enantiomerization or linearization significantly impaired the ability of HNP1 and HD5 to kill Staphylococcus aureus but not Escherichia coli. In contrast, LHNP4 and DHNP4 were equally bactericidal against both bacteria. d-Enantiomers were generally weaker inhibitors or binders of lethal factor and gp120 than their respective native, all-l forms, although activity differences were modest, particularly for HNP4. A strong correlation existed among these different functions. Our data indicate: (a) that HNP1 and HD5 kill E. coli by a process that is mechanistically distinct from their actions that kill S. aureus and (b) that chiral molecular recognition is not a stringent prerequisite for other functions of these defensins, including their ability to inhibit lethal factor and bind gp120 of HIV-1.

Human neutrophil α-defensin 4 inhibits HIV-1 infection in vitro

Human neutrophil α‐defensin 4 (HNP4) is more effective than HNP1–3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV‐1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV‐1 inhibition by HNP4 stems at least partially from a unique and lectin‐independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti‐HIV‐1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.

Reconstruction of the conserved beta-bulge in mammalian defensins using D-amino acids.

Defensins are cationic antimicrobial mini-proteins that play important roles in the innate immune defense against microbial infection. Six invariant Cys residues in each defensin form three structurally indispensable intramolecular disulfide bridges. The only other residue invariant in all known mammalian defensins is a Gly. Structural studies indicate that the invariant Gly residue is located in an atypical, classic-type β-bulge with the backbone torsion angles (ϕ, Ψ) disallowed for l-amino acids but permissible for d-enantiomers. We replaced the invariant Gly17 residue in human neutrophil α-defensin 2 (HNP2) by l-Ala or one of the d-amino acids Ala, Glu, Phe, Arg, Thr, Val, or Tyr. Although l-Ala17-HNP2 could not be folded, resulting in massive aggregation, all of the d-amino acid-substituted analogs folded with high efficiency. The high resolution x-ray crystal structures of dimeric d-Ala17-HNP2 were determined in three different crystal forms, showing a well preserved β-bulge identical to those found in other defensins. The seven d-analogs of HNP2 exhibited highly variable bactericidal activity against Gram-positive and Gram-negative test strains, consistent with the premise that interplay between charge and hydrophobicity dictates how amphiphilic defensins kill. Further, the bactericidal activity of these d-amino acid analogs of HNP2 correlated well with their ability to induce leakage from large unilamellar vesicles, supporting membrane permeabilization as the lethal event in microbial killing by HNP2. Our findings identify a conformational prerequisite in the β-bulge of defensins essential for correct folding and native structure, thereby explaining the molecular basis of the Gly-Xaa-Cys motif conserved in all mammalian defensins.

Why Is the Arg5-Glu13 Salt Bridge Conserved in Mammalian α-Defensins?

Mammalian α-defensins, expressed primarily in leukocytes and epithelia, kill a broad range of microbes, constituting one of the first lines of innate immune defense against infection. Nine amino acid residues, including six cysteines, one glycine, and a pair of oppositely charged residues Arg/Glu, are conserved in the otherwise diverse sequences of all known mammalian α-defensins. Structural analysis indicates that the two charged residues form a salt bridge, likely stabilizing a protruding loop in the molecule. To investigate the structural and functional roles of the conserved Arg5-Glu13 salt bridge in α-defensins, we chemically prepared human neutrophil α-defensin 2 (HNP2) and five HNP2 analogs, R5E/E13R, E13Q, E13R, R5T/E13Y, and R14A. In contrast to HNP2 and R14A-HNP2, none of the four salt bridge analogs was capable of folding into a native conformation in the context of isolated defensin domains. However, when covalently attached to the 45-residue pro-HNP2 propeptide, the salt bridge analogs of HNP2 in their pro-forms all folded productively, suggesting that the Arg5-Glu13 salt bridge is not required for correct pro-α-defensin folding. When assayed against both Escherichia coli and Staphylococcus aureus, the six α-defensins showed bactericidal activity that correlated with the number of net positive charges carried by individual molecules in the panel, irrespective of whether or not the Arg5-Glu13 salt bridge was decimated, suggesting that Arg5 and Glu13 are not functionally conserved. Proteolytic resistance analysis with human neutrophil elastase, one major protease contained in azurophils with HNPs, revealed that destabilization of the salt bridge dramatically accelerated defensin degradation by the enzyme. Thus, we propose that the Arg5-Glu13 salt bridge found in most mammalian α-defensins is conserved for defensin in vivo stability.

Functional Determinants of Human Enteric α-Defensin HD5 CRUCIAL ROLE FOR HYDROPHOBICITY AT DIMER INTERFACE

Background: Human α-defensin HD5 is a multifunctional antimicrobial peptide whose functional determinants have yet to be elucidated. Results: Alanine scanning mutagenesis aided by x-ray crystallography identified Leu29 at the dimer interface as crucial; N-methylation of Glu21 to debilitate HD5 dimerization also affected activity. Conclusion: Dimerization and hydrophobicity are important for HD5 function. Significance: The molecular basis of α-defensin function is better understood. Human α-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins, such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human α-defensin family: four human neutrophil peptides, including HNP1, and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However, the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine (Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel β strands. The effect of mutating Leu29 is similar to that of a C-terminal hydrophobic residue of HNP1, Trp26. In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by N-methylation of the Glu21 residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the α-defensins, revealing a crucial role of hydrophobicity-mediated dimerization.

Sometimes It Takes Two to Tango CONTRIBUTIONS OF DIMERIZATION TO FUNCTIONS OF HUMAN α-DEFENSIN HNP1 PEPTIDE

Background: Human α-defensin 1 (HNP1) is a small but functionally versatile antimicrobial peptide that exists as dimers and oligomers. Results: Destabilization of HNP1 dimer significantly impairs its ability to kill S. aureus, inhibit anthrax lethal factor, and bind HIV-1 gp120. Conclusion: Dimerization and oligomerization are important for many activities of HNP1. Significance: The molecular basis of functional versatility of human α-defensins is better understood. Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.

Structural and Functional Analysis of the Pro-Domain of Human Cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

Invariant Gly Residue Is Important for α-Defensin Folding, Dimerization, and Function A CASE STUDY OF THE HUMAN NEUTROPHIL α-DEFENSIN HNP1

Background: The conservation of the only invariant noncysteine residue in the α-defensins, Gly17, is poorly understood. Results: The G17A mutation impaired dimerization of the human α-defensin HNP1 and its ability to self-associate, inhibit anthrax lethal factor, and kill bacteria. Conclusion: Gly17 is totally conserved for both structural and functional reasons. Significance: The molecular determinants of human α-defensin function are better understood. The human α-defensins (HNP) are synthesized in vivo as inactive prodefensins, and contain a conserved glycine, Gly17, which is part of a β-bulge structure. It had previously been shown that the glycine main chain torsion angles are in a d-configuration, and that d-amino acids but not l-alanine could be substituted at that position to yield correctly folded peptides without the help of a prodomain. In this study, the glycine to l-alanine mutant defensin was synthesized in the form of a prodefensin using native chemical ligation. The ligation product folded correctly and yielded an active peptide upon CNBr cleavage. The l-Ala17-HNP1 crystal structure depicted a β-bulge identical to wild-type HNP1. However, dimerization was perturbed, causing one monomer to tilt with respect to the other in a dimerization model. Inhibitory activity against the anthrax lethal factor showed a 2-fold reduction relative to wild-type HNP1 as measured by the inhibitory concentration IC50. Self-association was slightly reduced, as detected by surface plasmon resonance measurements. According to the results of the virtual colony count assay, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus exhibited a less than 2-fold reduction in virtual lethal dose values. Prodefensins with two other l-amino acid substitutions, Arg and Phe, at the same position did not fold, indicating that only small side chains are tolerable. These results further elucidate the factors governing the region of the β-bulge structure that includes Gly17, illuminating why glycine is conserved in all mammalian α-defensins.

Effects of the terminal charges in human neutrophil α-defensin 2 on its bactericidal and membrane activity

Human neutrophil alpha-defensin 2 (HNP2) was N-terminally acetylated and/or C-terminally amidated, resulting in three terminally modified analogs, Ac-HNP2, HNP2-NH2 and Ac-HNP2-NH2. We examined their bactericidal activity against E. coli and S. aureus and their ability to induce leakage from large unilamellar vesicles. Loss of the N-terminal positive charge was functionally deleterious, whereas removal of the C-terminal negative charge enhanced microbial killing and membrane permeabilization. Our findings validate the importance of electrostatic forces in defensin-microbe interactions and point to the bacterial cytoplasmic membrane as a target of HNP2 activity.