Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA

Indolicidin, a l3-residue antimicrobial peptide-amide, which is unusually rich in tryptophan and proline, is isolated from the cytoplasmic granules of bovine neutrophils. In this study, the structures of indolicidin in 50% D₃-trifluoroethanol and in the absence and presence of SDS and D₃₈-dodecylphosphocholine were determined using NMR spectroscopy. Multiple conformations were found and were shown to be due to different combinations of contact between the two WPW motifs. Although indolicidin is bactericidal and able to permeabilize bacterial membranes, it does not lead to cell wall lysis, showing that there is more than one mechanism of antimicrobial action. The structure of indolicidin in aqueous solution was a globular and amphipathic conformation, differing from the wedge shape adopted in lipid micelles, and these two structures were predicted to have different functions. Indolicidin, which is known to inhibit DNA synthesis and induce filamentation of bacteria, was shown to bind DNA in gel retardation and fluorescence quenching experiments. Further investigations using surface plasmon resonance confirmed the DNA-binding ability and showed the sequence preference of indolicidin. Based on our biophysical studies and previous results, we present a diagram illustrating the DNA-binding mechanism of the antimicrobial action of indolicidin and explaining the roles of the peptide when interacting with lipid bilayers at different concentrations.     

New indolicidin analogues with potent antibacterial activity.

Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe(4,6,8,9,11)] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.     

Serial Expression and Activity Analysis of LNK-16: A Bovine Antimicrobial Peptide Analogue

Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)₃ in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)₃ was purified by Ni²⁺-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.     

Antimicrobial activity of the indolicidin‐derived novel synthetic peptide In‐58

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin‐derived novel synthetic peptide In‐58. In‐58 was generated by replacing all tryptophan residues on phenylalanine in D‐configuration; the α‐amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In‐58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In‐58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD’::lux), we investigated the action of indolicidin and In‐58 at the subcellular level. At subinhibitory concentrations, indolicidin and In‐58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Bacterial selectivity and plausible mode of antibacterial action of designed Pro-rich short model antimicrobial peptides.

To develop novel Pro-rich model AMPs with shorter length and higher bacterial selectivity/therapeutic index (TI) than natural AMP, indolicidin, we synthesized a series of undodecapeptides derived from the sequence XXPXXPWXPXX-NH2 (X indicates Leu or Lys) with different ratios of Lys and Leu residues. Several Pro-rich model peptides (K7 WP3, K6 WL1 P3, K5 WL2 P3-1, K5 WL2 P3-2, and K4 WL3 P3) had approximate 8- to 11-fold higher bacterial selectivity/TI compared to indolicidin. These peptides selectively bind to negatively charged liposomes (EYPG/EYPG; 7:3, w/w) mimicking bacterial membranes. Their high selectivity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Indolicidin showed almost complete depolarization of the cytoplasmic membrane of Staphylococcus aureus and dye-leakage from negatively charged liposomes at 10 microM, whereas all of Pro-rich model peptides had very little activity in these assays even at 80 microM, as observed in buforin 2. These results suggest that the ultimate target of our designed Pro-rich model peptides is probably the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed Pro-rich short model peptides appear to be excellent candidates for future development as a novel antimicrobial agent.     

E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (k_obs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of k_obs on TDG concentration gives k_max = 1.6 min⁻¹ and K_1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (K_d ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.     

Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination

The Streptomyces phage C31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5′ ends. Rotation of one pair of half sites by 180° relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by C31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.     

Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

5-fluorouracil (5-FU) is a widely used anticancer drug that disrupts pyrimidine nucleotide pool balances and leads to uracil incorporation in DNA, which is then recognized and removed by the uracil base excision repair (BER) pathway. Using complementary biochemical and genetic approaches we have examined the role of uracil BER in the cell killing mechanism of 5-FU. A yeast strain lacking the enzyme uracil DNA glycosylase (Ung1), which excises uracil from the DNA backbone leaving an abasic site, showed significant protection against the toxic effects of 5-FU, a G₁/S cell cycle arrest phenotype, and accumulated massive amounts of U/A base pairs in its genome (~4% of T/A pairs were now U/A). A strain lacking the major abasic site endonuclease of Saccharomyces cerevisiae (Apn1) showed significantly increased sensitivity to 5-FU with G₂/M arrest. Thus, efficient processing of abasic sites by this enzyme is protective against the toxic effects of 5-FU. However, contrary to expectations, the Apn1 deficient strain did not accumulate intact abasic sites, indicating that another repair pathway attempts to process these sites in the absence Apn1, but that this process has catastrophic effects on genome integrity. These findings suggest that new strategies for chemical intervention targeting BER could enhance the effectiveness of this widely used anticancer drug.     

Formation and characterization of a single Trp-Trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity

Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and beta-structured cationic peptides, because five of indolicidin's 13 residues are tryptophans: H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH(2). Solid phase synthesis of indolicidin gave rise to a minor byproduct that possessed unusual fluorescence and UV absorbance properties compared with authentic indolicidin. The byproduct was purified by combined ion exchange and reversed phase high pressure liquid chromatography steps and was shown be identical to authentic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass analysis of the byproduct revealed a 2-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to form an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed the nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edman degradation revealed that Trp-6 and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digestion with trypsin and chymotrypsin, suggesting that the ditryptophan stabilizes a subset of molecular conformations that are protease resistant but that are absent in the native structure.     

CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix.

Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.     

Characterization of the Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg²⁺, while ssDNA AP site cleavage required Mg²⁺ (optimally at 0.5-2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3′-PO₄²⁻ group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.     

Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.     

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP₊₃-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP₊₃-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′ → 5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.     

Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties,cytotoxicity and mechanism of action

Animals posses a large variety of antimicrobial peptides (AMPs) that serve as effective components in innate host defenses against microbial infections. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. This study was designed to evaluate their therapeutic potential of the following six antimicrobial peptides initially found from animals: cecropin P1, indolicidin, LL-37, palustrin-OG1, LFP-20 and LFB-11. Our results indicated that cecropin P1 possessed the most desired biological activity, with fast and potent antimicrobial activity but only slight hemolytic or cytotoxic activity against human cells. Indolicidin was more effective against gram-positive bacteria but with higher hemolytic and cytotoxic activity on human peripheral blood mononuclear cell (PBMCs) (P < 0.05). Although LFP-20 and LFB-11 had moderate activity against tested strains and need 30 min to kill E. coli, they showed almost no hemolytic and cytotoxic activity towards PBMCs (P < 0.01). Indolicidin could form pores of well-defined structure in bacterial membranes whereas lysis of E. coli cells was observed after addition LFB-11 and LL-37 at 1 × MIC for 1 h. LL-37 treatment could lead to the leakage of entire bacterial cytoplasmic contents. The most obvious phenomenon was protuberant structures on the E. coli cell surface after incubation with LFP-20, cecropin P1 and palustrin-OG1. The results presented here illustrate that AMPs derived from different animals exhibited different antimicrobial characteristics. Because of their potent and broad-spectrum antimicrobial activity, low cytotoxicity towards normal cells, and the unique mechanism of action, these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.     

Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase

HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D₆₄, D₁₁₆ and E₁₅₂ of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V₁₅₁E₁₅₂S₁₅₃ region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3′-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V₁₅₁E₁₅₂S₁₅₃ region involvement in the integration reaction is more important than for the 3′-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A20_1–50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A20_1–50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a K_d of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.