Erik de Leeuw

Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II

Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human α‐defensins does not correlate with antibacterial killing. We further show that the α‐defensin human neutrophil peptide‐1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.

Toward Understanding the Cationicity of Defensins ARG AND LYS VERSUS THEIR NONCODED ANALOGS

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in α-defensins but not in β-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human α-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six α-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys (diMeLys), and homo-Arg (homoArg). In addition, we prepared human β-defensin 1 (hBD1) and Lys→ArghBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and Lys→ArghBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the α-defensin than in the β-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.

Molecular characterization of Escherichia coli FtsE and FtsX.

The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP‐binding cassette (ABC)‐type transporter. FtsE is a hydrophilic nucleotide‐binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.

Membrane association of FtsY, the E. coli SRP receptor

FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SRα), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SRα, FtsY consists of two major domains: a strongly acidic N‐terminal domain (A) and a C‐terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associate with the membrane but that the nature of the association differs.

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.

Trp-26 imparts functional versatility to human alpha-defensin HNP1.

We performed a comprehensive alanine scan of human α-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

Selective arginines are important for the antibacterial activity and host cell interaction of human α-defensin 5

Defensins constitute a major family of natural antimicrobial peptides that protect the host against microbial invasion. Here, we report on the antibacterial properties and cellular interaction of Human Defensin 5 as a function of its positive charge and hydrophobicity. We find that selective replacement of arginine residues in HD‐5 by alanine or charge‐neutral lysine residues reduces antibacterial killing as well as host cell interaction. We identify arginines at positions 9 and 28 in the HD‐5 sequence as particularly important for its function. Replacement of arginine at position 13 to Histidine, as observed in a Crohns disease patient, reduced bacterial killing strain‐selectively. Finally, we find that HD‐5 interacts with host cells via receptor‐mediated mechanisms.

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.

The Conserved Salt Bridge in Human α-Defensin 5 Is Required for Its Precursor Processing and Proteolytic Stability

Mammalian α-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 β-bulge are totally conserved in the otherwise diverse sequences of all known mammalian α-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 Arg6–Glu14 salt bridge in human α-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 Arg13–Gln14 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 α-defensin in vivo.

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.