Báo cáo khoa học: Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin pot

Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin Goran Kragol 1 , Ralf Hoffmann 2 , Michael A. Chattergoon 1 , Sandor Lovas 3 , Mare Cudic 1 , Philippe Bulet 4 , Barry A. Condie 1 , K. Johan Rosengren 5 , Luis J. Montaner 1 and Laszlo Otvos Jr 1 1 The Wistar Institute, Philadelphia, PA, USA; 2 Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universita ¨ t, Du ¨ sseldorf, Germany; 3 Department of Biomedical Sciences, School of Medicine, Creighton University, Omaha, NB, USA; 4 Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France; 5 Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure–antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2–10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluo- rescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin signi- ficantly reduced the peptide’s ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin’s suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocori- cin analogs modified at strategic positions, we further veri- fied that DnaK was the bacterial target macromolecule. In addition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correla- ted. Mutational analysis on a synthetic E. coli DnaK frag- ment identified a possible binding surface for pyrrhocoricin. Keywords: antimicrobial peptides; cell penetration; heat shock proteins; mutational analysis; pharmacophore. In the desperate fight against antibacterial-resistant bacteria, the proline-rich peptide family can come to the rescue [1]. The most active member of these peptides, pyrrhocoricin, is nontoxic to healthy mice and eukaryotic cells, and at low doses can protect mice from bacterial challenge [2]. Because at high doses pyrrhocoricin is toxic to compromised animals, pyrrhocoricin analogs were developed that lack in vivo toxicity, even in infected mice, and show improved stability in mammalian sera [2,3]. These designed peptides efficiently kill various resistant strains and clinical isolates [4]. The key feature to the ability of these peptides to kill resistant bacteria is their novel mechanism of action. Pyrrhocoricin, and other family members such as drosocin and apidaecin appear to exhibit their antimicrobial activity by binding to the bacterial heat shock protein DnaK [5], preventing chaperone assisted protein folding and inhibiting the strongly related ATPase activity of DnaK [6]. Similar to other antimicrobial peptides [7], and generally peptides that penetrate mammalian cells [8], the proline-rich peptides are also rich in cationic residues, arginine, lysine and histidine. Cationic peptides interact electrostatically with the negatively charged bacterial phospholipids and then insert into the model membranes of planar bilayers or liposomes [9,10]. Although these processes often involve the formation of channels or pores, alternative mechanisms were also suggested that either cooperatively destroy the membrane barrier without channel formation [11] or just create brief disruptions in permeability [12]. It is clear that permeabilization of the cytoplasmic membrane to destroy the membrane potential is not lethal per se [13]. This is especially true for peptides that are rich not only in positively charged residues but prolines as well [14]. For example, it was argued that apidaecin or its mammalian analog PR-39, enter bacterial cells without any disruption of the membrane structure, and consecutively meet their intracellular target biopolymer [15,16]. The special role of proline residues has been best documented for buforin II, an antimicrobial peptide acting on bacterial DNA and RNA [17]. Confocal fluorescence microscopy studies show that buforin II analogs with an intact proline hinge penetrate Escherichia coli membranes without permeabili- zation and accumulate in the cytoplasm [18]. When the key proline is replaced with leucine, the preferred amino acid residue in antibacterial peptides with amphipathic a-helical structure [19], the ability of the peptide to accumulate intracellularly is decreased, and therefore the antibacterial activity is reduced. The above results suggest that the DnaK-binding domain of pyrrhocoricin may not span the entire molecule, and a shorter active site can be identified that might not share the Correspondence to L. Otvos, Jr., The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. Fax: + 1 215 898 5821, Tel.: + 1 215 898 3772, E-mail: Otvos@wistar.upenn.edu (Received 24 May 2002, revised 2 July 2002, accepted 11 July 2002) Eur. J. Biochem. 269, 4226–4237 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03119.x sequence with the fragment responsible for bacterial or eukaryotic cell entry. Therefore, we studied the antibacterial activity of a series of pyrrhocoricin analogs, in which either each residue was replaced with alanine (Ala-scan) or arginine-containing modules were inserted into strategically important positions in the peptide. We also wanted to know whether a pyrrhocoricin-based delivery mechanism could promote entry into the less permeable mammalian cells [20]. When we ascertained that the Asp2–Pro10 fragment is the most crucial domain for killing of E. coli and Agrobacte- rium tumefaciens, we further investigated the interaction between fluorescein-labeled pyrrhocoricin or its active or inactive analogs and E. coli DnaK. In another line of investigation, four pyrrhocoricin-responsive and seven unresponsive bacterial and fungal strains were selected and pyrrhocoricin’s binding to the corresponding DnaK D-E helix was studied by fluorescence polarization. The anti- bacterial activity correlated strongly with the binding to the multihelical lid region of DnaK. Finally an Ala-scan on the synthetic E. coli DnaK D-E helix fragment identified a series of residues that could not be replaced without losing binding to pyrrhocoricin. In the crystal structure of the DnaK protein, these residues comprise a continuous binding surface. MATERIALS AND METHODS Rationale for the design of the modified pyrrhocoricin analogs Modified pyrrhocoricin analogs were designed for studying important residues and regions in the hypothetical active site and delivery module (Table 1). First we concentrated on the pharmacophore. In peptide Pyrr-mod1, a characteristic turn-forming Pro8-Arg9 dipeptide motif in the active site was replaced with helix-forming residues Glu and Ala. To maintain the overall number of positive charges, the apparently freely replaceable Ser5 was substituted with Arg. The same design principles were applied to peptide Pyrr-mod2 with an additional positive charge at the Table 1. Synthetic peptides used in this study. Where appropriate, gaps are inserted to help the comparison of the different peptide sequences. Unlabeled pyrrhocoricin analogs Pyrrhocoricin VDKG S YL P R PT P PR P IY NR N Pyrr-mod1 VDKG R YL E A PT P PR P IY NR N Pyrr-mod2 DKG RYL EAPTR PRP ERNRK Pyrr-mod3 VDKG S YL P R PT P Y S PR P P IY NR N Pyrr-mod4 VDKG S YL P R PT P T PR Y RP IY NR N Fluorescein-labeled full-sized pyrrhocoricin derivatives a Fl-K-pyrrhocoricin 1-20 Fluorescein-K-VDKGSYLPRPTPPRPIYNRN Fl-Pyrr-mod1 Fluorescein- VDKGRYLEAPTPPRPIYNRN Fl-Pyrr-mod2 Fluorescein- VDKGRYLEAPTRPRPERNRK Fl-Pyrr-mod3 Fluorescein- VDKGSYLPRPTPYSPRPPIYNRN Fl-Pyrr-mod4 Fluorescein- VDKGSYLPRPTPTPRYRPIYNRN Fl-K-Pyrr-mod4 Fluorescein-K-VDKGSYLPRPTPTPRYRPIYNRN Pyrrhocoricin fragments (unlabeled and labeled) b K-Pyrr-1-10 K-VDKGSYLPRP Fl-K-pyrrhocoricin 1-10 Fluorescein-K-VDKGSYLPRP Fl-K-pyrrhocoricin 1-9 Fluorescein-K-VDKGSYLPR Fl-K-pyrrhocoricin 11-20 Fluorescein-K-TPPRPIYNRN Pyrr-10-20-K(Fl) PTPPRPIYNRN-K(Fluorescein) DnaK and Hsp70 D-E helix fragments: Escherichia coli 583-615 IEAKMQELAQVSQKLMEIAQQQHAQQQTAGADA Agrobacterium tumefaciens 581-614 IQAKTQTLMEVSMKLGQAIYEAQQAEAGDASAEG Haemophilus influenzae 583-615 IEAKIEAVIKASEPLMQAVQAKAQQAGGEQPQQ Haemophilus ducreyi 583-615 IEAKIEAVIKASEPLMQAAQAKAQTNQAGEQQS Helicobacter pylori 578-607 AELEDKTKLLAQAAQKLGEAMANKNNAEQP Pseudomonas aeruginosa 584-615 IEAKMNALSQASTPLAQKMYAEQAQQGEDAPQ Staphylococcus aureus 554-585 IKSKKEELEKVIQELSAKVYEQAAQQQQQAQG Streptococcus pyogenes 554-588 MKAKLEALNEKAQALAVKMYEQAAAAQQAAQGAEG Streptococcus pneumoniae 554-588 MKAKLEALNEKAQQLAVKLYEQAAAAQQAQEGAEG Candida albicans 591-623 YEDKRKELESVANPIISGAYGAAGGAPGGAGGF Mus musculus 595-626 (Hsp70-2) YEHKQKELERVCNPIISKLYQGGPGG Homo sapiens 592-624 (Hsp70-1) FEHKRKELEQVCNPIISGLYQGAGGPGPGGFGA a The fluorescein-label was directly coupled to the amino-termini of the peptides. b The fluorescein-label was directly coupled to the amino- termini of the peptides, except for the Pyrr-10-20-K(Fl) derivative in which the fluorescein was coupled to the side chain of the extra C-terminal lysine residue. The Fl-K-pyrrhocoricin 1-9 and Fl-K-pyrrhocoricin 1-10 peptides behaved very similarly in every experiment, and therefore data are provided only for the 1-9 analog that had been scrutinized for its ATPase inhibitory activity earlier [6]. The fluorescence signal of the two C-terminal analogs was quenched very rapidly. They are part of our ongoing efforts to find a suitable carboxy-terminal substrate. Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur. J. Biochem. 269) 4227 C-terminus to further promote cell entry. In addition, the nonessential and freely replaceable Ile-Tyr dipeptide was substituted with the Glu-Arg dipeptide to maintain good solubility at any pH. Later we made changes only in the C-terminal, nonessential region. In peptide Pyrr-mod3, while the active site was kept intact, the characteristic Pro12- Pro13 dipeptide fragment of the C-terminal domain was disrupted, by inserting two hydroxy-amino acids that are not foreign to pyrrhocoricin, but are normally located outside the Pro-Arg-Pro repeats. To compensate for the reduction of the percentage of Pro, an additional proline was inserted after the last Pro, before the final IYNRN segment. Finally, in peptide Pyrr-mod4 the same design principles were used, except the inserted two hydroxy-amino acids were separated, and the positive charge of the putative cell-penetrating region was increased by inserting an addi- tional Arg residue. N-Terminally fluorescein-labeled peptides included full- sized native pyrrhocoricin, apidaecin and drosocin [6], and the N-terminal 1–9 fragment of pyrrhocoricin. In these derivatives, 5(6)-carboxy-fluorescein was coupled to the a-amino group of an extra N-terminal lysine residue. The addition of this Lys was needed, because the antibacterial activity of the pyrrhocoricins is lost upon directly blocking the N-terminus without an extra positive charge ([2] and Table 2). This positive charge apparently plays a role in the interaction with bacterial membranes [21]. The Pyrr-mod1– 4 peptides were also synthesized with an N-terminal fluorescein label, but because these peptides were designed for fluorescence polarization studies, they did not contain the extra lysine, except peptide Fl-Pyrr-mod4, which was made both with and without the N-terminal Lys. In support of the earlier findings, the peptide without the lysine lost its antibacterial activity (Table 2). Table 2 also demonstrates that the addition of the N-terminal lysine did not turn an otherwise inactive peptide into an active one against E. coli JC7623. A series of peptides corresponding to the D-E helix regions of different bacterial and eukaryotic DnaK/Hsp70 sequences were synthesized. The different Hsp70 sequences were dissimilar towards to C-termini of the proteins. The decision as to which residues to select as C-termini depended upon where a gap between the extended D-E helix and the short extreme C-terminal domain was found. We observed the earliest gaps for the H. pylori and the mouse sequences. To have bacterial DnaK fragments of similar size, the H. pylori peptide was extended by two residues toward the D-helix at the N-terminus. The Salmonella typhimurium DnaK 584– 616 fragment has not been prepared because it is essentially identical to the E. coli sequence except for a conservative TAG fi AGS change very close to the C-terminus. For the Ala-scan of the DnaK D-E helix region, all native residues were replaced with alanine. The three native alanines, Ala591, Ala601 and Ala606 were replaced with phenylalanine. The sequence was not continued after Thr610 because the C-terminal hydrophobic residues signi- ficantly contribute to the low aqueous solubility of the full 583–610 fragment. Peptide synthesis and purification N-Terminally Fmoc-protected amino acids [22] were used for the synthesis of the peptides. In the Ala-scans each residue in pyrrhocoricin or each residue in the 583–610 E. coli DnaK fragment was replaced with alanine. The Ala- scan peptides were assembled on a SYRO multiple peptide synthesizer, and after cleavage were purified by RP-HPLC. All other peptides were made individually, on a MilliGen 9050 continuous-flow automated synthesizer and were purified by RP-HPLC until MALDI-MS revealed only single species with the expected molecular ions. The mass spectra were taken at the Wistar Institute Protein Micro- chemistry Facility. Fluorescence polarization Binding of the synthetic DnaK fragments or the mutated derivatives to fluorescein-labeled pyrrhocoricin or its ana- logs was assessed by fluorescence polarization [23]. For these experiments, the unlabeled peptides were serially diluted in Tris buffer (pH 7.4) in 50 lL final volume in 6 · 50 mm disposable glass borosilicate tubes. The fluores- ceinated pyrrhocoricin peptides were added to each tube in a 50-lL aliquot to a final concentration of 1 n M and tubes were incubated at 37 °C for 5 min The extent of fluores- cence anisotropy was measured on a Beacon 2000 fluores- cence polarization instrument (PanVera, Madison, WI, USA) and calculated as millipolarization values. The filters used were 485 nm excitation and 535 nm emission with 3 nm band width. During fluorescence polarization, it is assumed that one molecule of the labeled compound interacts with one molecule of the unlabeled partner, and while the concentration of the unlabeled larger molecule determines the association constant, the concentration of the labeled partner determines the fluorescence intensity. The 1 n M tracer concentration, andother parameters used herein, were successfully used in our laboratory earlier to study peptide–peptide [6,24], peptide–protein [5] and pep- tide–nucleic acid interactions [25]. In vitro antibacterial assays Antibacterial assays were performed in sterile 96-well plates (Nunc F96 microtiter plates) with a final volume of 100 lL Table 2. In vitro antibacterial activity of some pyrrhocoricin analogs that are discussed in the text. In addition to the fluorescein-labeled Pyrr- mod1 – Pyrr-mod4 pyrrhocoricin derivatives, the same peptide analogs were labeled with biotin at their N-termini. Similar to the fluorescein- derivatives, none of the biotin-labeled peptides exhibited activity up to 40 l M . Peptide IC 50 against E. coli JC7623 (l M ) L -pyrrhocoricin < 0.3 D -pyrrhocoricin > 40 L -pyrrhocoricin-1-10- D -pyrrhocoricin-11-20 > 40 Fl-K-pyrrhocoricin 1-20 20–40 Fl-Pyrr-mod1 > 40 Fl-Pyrr-mod2 > 40 Fl-Pyrr-mod3 > 40 Fl-Pyrr-mod4 > 40 Fl-K-Pyrr-mod4 10–20 K-pyrr-1-10 > 40 4228 G. Kragol et al. (Eur. J. Biochem. 269) Ó FEBS 2002 as described previously [26]. Briefly, 90 lL of a suspension of a mid-logarithmic phase bacterial culture at an initial 600 nm UV absorbance of 0.001 in media required for good growth of the various bacterial strains was added to 10 lL of serially diluted peptides in sterilized water. The final peptide concentrations ranged between 0.06 l M (lower) and 20–80 l M (upper). Plates were incubated at 37 °Cfor 16–24 h with gentle shaking, and growth inhibition was measured by recording the increase of the UV absorbance at 600 nm on a microplate reader. Cell penetration assay To study the ability of the antibacterial peptides and their fragments to enter cells, fluorescein-labeled peptides were added to E. coli JC7623 cells or mouse macrophages at final concentrations of 5 lgÆmL )1 and 100 ngÆmL )1 , respectively. The cells were allowed to acquire the substrate for 60 min at 37 °C, the excess substrate was removed and the cells were washed extensively with NaCl/P i , pH 6.8. The cells were fixed with NaCl/P i buffer containing 1% paraformaldehyde and visualized using a Leica TCS SPII laser scanning confocal microscope (E. coli) or Leica DMIL fluorescence microscope (macrophages). Circular dichroism measurements CD spectra were performed on a Jasco J720 instrument at room temperature in a 0.2 mm pathlength cell. Doubly distilled water was used as solvent. The peptide concentra- tions were  0.5 mgÆmL )1 , determined each time by quan- titative RP-HPLC [27]. Curves were smoothed by the algorithm provided by Jasco. Mean residue ellipticity ([h] MR ) is expressed in degreesÆcm 2 Ædmol )1 by using mean residue masses of 110. RESULTS Identification of crucial residues for the antibacterial activity of pyrrhocoricin An Ala-scan was performed to identify key residues that could not be replaced in pyrrhocoricin without a major loss in antibacterial activity. For the bacterial strains, we selected E. coli and A. tumefaciens because in our earlier studies we had observed some differences in the potency of truncated pyrrhocoricin peptides in killing these strains [28]. Accord- ing to the Ala-scan, pyrrhocoricin had an identical bioactive fragment against both strains (Fig. 1). No activity was detected (up to 20 l M peptide) against either bacterium whenAsp2,Lys3,Tyr6,Leu7,Pro8,Arg9orPro10were replaced with alanine, indicating that the most crucial residues for antibacterial activity were in the Asp2–Pro10 peptide fragment. Further investigations are required to reveal whether the Asp2–Lys3 dipeptide is a functional part of the pharmacophore. All other amino acids could be replaced without a major loss in the antimicrobial activity of the peptide, except Val1, Arg14 and Arg19, residues, which are needed for the full antibacterial activity, at least against A. tumefaciens. Some key N-terminal residues were also replaced with tyrosine (Tyr-scan), resulting in identical activity data. Antibacterial activity of pyrrhocoricin analogs The Ala- and Tyr-scans identified the N-terminus as the fragment carrying essential residues for the antibacterial activity and the C-terminus as a necessary domain, but suitable for substitutions. To provide further support for this notion, the antibacterial activity of the Pyrr-mod1– Pyrr-mod4 analogs was studied in detail against clinical strains of E. coli, A. tumefaciens, S. typhimurium, H. influ- enzae, Klebsiella pneumoniae, P. aeruginosa and S. aureus. Table 2 shows the half inhibitory concentration (IC 50 ) values against these bacteria. As we showed earlier, the pyrrhocoricin analogs are inactive against S. aureus [5], and no activity could be obtained with the new peptides, even if the number of the positive charges was increased. When modifications were made in the Tyr6–Pro10 active segment (peptides Pyrr-mod1 and Pyrr-mod2) the activity was completely lost against all five originally responsive strains. Most, but not all, activity could be recovered by peptide Pyrr-mod3, which had no modification in the Tyr6–Pro10 region. However in peptide Pyrr-mod4, a repeat of the characteristic Thr11–Pro12 linker between the active seg- ment and the C-terminal domain, coupled with the increase of the positive charges needed for cell entry, resulted in a peptide variant with increased antibacterial activity against two of the five strains. When we synthesized pyrrhocoricin from L -amino acid residues at the N-terminus (Val1-Pro10) and D -amino acid residues at the C-terminus (Thr11- Asn20), no activity against E. coli JC7623 was observed (Table 2). Antibacterial peptides with mainly membrane- disrupting mode of action are insensitive to the stereochem- ical configuration of the constituent residues [29]. Peptide entry into E. coli cells The experiments above indicated that the proline-rich peptide family had multiple functions and functional domains, and perhaps carried separate modules for cell entry and other tasks leading to bacterial killing. Therefore, we studied the ability of fluorescein-labeled pyrrhocoricin, its fragments and modified analogs to enter E. coli JC7623 cells. Because these peptides are currently considered promi- Fig. 1. In vitro antibacterial activity of alanine-substituted pyrrhocoricin peptides (Ala-scan) against Escherichia coli D22 and Agrobacterium tumefaciens. The data represent the IC 50 values, which were read as half ultraviolet absorbance between full bacterial growth (no peptide added) and medium. The assay was performed in poor broth medium. Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur. J. Biochem. 269) 4229 sing therapeutic drug candidates [1,3,4], and many emerging resistant bacterial species are intracellular pathogens, we also wanted to know whether the proline-rich antibacterial peptide family was able to penetrate macrophages where facultative or obligate bacteria often propagate. N-Terminally labeled pyrrhocoricin, containing a lysine residue between the label and the antibacterial peptide (Fl-K-pyrrhocoricin 1–20), entered the bacterial cells very efficiently (Fig. 2, top left image). The staining appeared to be homogenous throughout the entire cell body. Although the isolated 1–9 fragment of pyrrhocoricin is inactive in the in vitro antibacterial efficacy test [2], this fragment binds to the D-E helix region of bacterial DnaK and inhibits the ATPase activity of the heat shock protein [6]. We wanted to see whether the lack of antibacterial activity could be correlated with less efficient cell entry. Indeed, much weaker labeling of the E. coli cells was observed for the N-terminal 1–9 fragment (Fl-K-pyrrhocoricin 1–9), carrying the fluorescein chromophore and the extra lysine residue in a position identical to that of full-sized pyrrhocoricin (Fig. 2, top right image). Not only fewer cells were labeled with the shortened pyrrhocoricin fragment, but perhaps more importantly, the fluorescence intensity seemed to concen- trate to the cell surface as the staining pattern of single cells indicated (Fig. 2, middle images). Widespread distribution of the drug inside bacterial cells is needed for efficient inhibition of the putative intracellular target protein. When a similarly labeled construct of Pyrr-mod4 was studied (Fl-K-Pyrr-mod4), the bacterial cell penetration profile was not significantly different from that of full-sized pyrrhoco- ricin, indicating that the function of the C-terminal module was not compromised with the amino acid changes and insertions in this domain. The removal of the Lys residue that connected the fluorescein moiety and Val1 of the antibacterial peptide analog resulted in a major decrease in the number of E. coli cells labeled by Fl-Pyrr-mod4, consistent with the significant reduction in the antibacterial activity (Table 2). Nevertheless, when Fl-Pyrr-mod4 with- out the extra lysine did enter bacterial cells, the peptide analog was homogenously distributed all over the cell interior (Fig. 2, bottom right image). Fl-Pyrr-mod1 without the extra lysine entered similar number of cells and underwent intracellular distribution in an identical fashion (Fig. 2., bottom left image), indicating that there were no major differences in the cell penetration and distribution pattern of naked Pyrr-mod1 or Pyrr-mod4. Rather, the considerably stronger antibacterial activity of naked Pyrr- mod4 (Table 3) was due to its actions on the intracellular target molecule. In addition to bacterial cells, fluorescein- labeled analogs of the three native peptides with the Lys Fig. 2. Confocal fluorescence microscopic images of E. coli JC7623 cells upon incubation with fluorescein-labeled pyrrhocoricin analogs. The peptide substrates are as follows: Fl-K- pyrrhocoricin 1–20, top and middle left; the analogous N-terminal half, Fl-K-pyrrho- coricin 1–9, top and middle right; the Fl-Pyrr- mod1 peptide without a Lys between the fluorescein label and the peptide analog, bottom left; and a similarly built Fl-Pyrr- mod4 construct, bottom right. 4230 G. Kragol et al. (Eur. J. Biochem. 269) Ó FEBS 2002 inserted at the N-terminus between the label and the peptides were added to murine macrophage cultures. Pyrrhocoricin, apidaecin and drosocin penetrated into mammalian cells without having preference to any partic- ular cell compartment, including the cell membrane (not shown). Similar to the results observed on bacterial cells, the identically labeled pyrrhocoricin 1–9 fragment penetrated into the mouse macrophages much less effectively. This also confirmed that while the N-terminus served as the pharma- cophore and hence the interactive domain with the bacterial target protein, the C-terminal half aided the delivery of the pharmacophore into the interior of cells. These studies require a detailed analysis of the permis- sibility of fluorescein substitutions at various positions in the peptide. Addition of fluorescein significantly reduces the antibacterial activity of pyrrhocoricin, much more than any other substitution does ([2] and Table 2), even if the Lys residue is incorporated between the label and the peptide. Therefore, the current cell penetration studies were restric- ted to peptides that could be labeled with fluorescein at the original N-termini in an identical manner. The fluorescence intensity of C-terminally labeled pyrrhocoricin fragments quenched very rapidly (compare with Table 1). In addition, the extra lysine residue alone did not generate antibacterial activity above that present without this addition. The K-Pyrr-1–10 peptide fragment was just as inactive against E. coli JC7623 as any other pyrrhocoricin fragments truncated to half size. Connection between antibacterial activity and pyrrhocoricin binding to the D-E helix region of DnaK Previously, we documented that pyrrhocoricin binds to a synthetic version of the D-E helix region of E. coli,a responsive species, but fails to bind to the homologous fragment of the unresponsive species S. aureus [6]. Here, we extended these studies to four responsive and seven unresponsive bacterial and fungal species. Table 3 shows that native pyrrhocoricin killed E. coli, A. tumefaciens, S. typhimurium,andH. influenzae, among others, and failed to kill S. aureus. Very weak activity, often read as inactive [3], was found against P. aeruginosa. Some other pyrrhocoricin-unresponsive bacterial and fungal strains include S. pyogenes, C. albicans [4], S.pneumoniae, H. duc- reyi and H. pylori. In addition, the peptide is not toxic to healthy mice or eukaryotic cells [2]. Fragments of the DnaK/Hsp70 sequences of the above-listed bacterial and fungal strains and of mouse and human were synthesized and their binding to N-terminally fluorescein-labeled pyr- rhocoricin (with the extra lysine inserted) was studied by fluorescence polarization. The synthetic DnaK peptides encompassed the D and E helices and a short fragment of the unordered region C-terminal to the multihelical lid assembly (Table 1). Because the corresponding sequences of E. coli and S. typhimurium DnaK are almost identical, and from other studies we know that the TAGADA C-terminal hexapeptide fragment of the E. coli sequence (where these minor mutations are found) is not needed for the binding, the S. typhimurium peptide was not synthesized. The K d between E. coli D-E DnaK and fluorescein-labeled pyrrho- coricin is in the order of 50 l M [6], is expected to be lower for the full-sized protein, and correlates well with the 5–10 l M antibacterial activity of the same fluorescein- labeled pyrrhocoricin against E. coli [2]. One set of the binding studies was carried out at a receptor fragment concentration of 15 l M , below the peptide–peptide associ- ation constant, serving the data as baseline values. The highest concentration of the synthetic DnaK fragment was 250 l M , where all responsive sequences were expected to bind. In summary, strong pyrrhocoricin binding to the D-E DnaK helix region of the three (four) responsive strains was observed, and no or very low binding was found to the DnaK fragments of the seven nonresponsive, or very weakly responsive strains (Fig. 3). Significantly, no binding was detected to the mouse or human Hsp70 fragment, confirm- ing the lack of specific binding to the human equivalent of DnaK [5]. While modifications in the active segment of pyrrhoco- ricin resulted in the complete disappearance of the antibac- terial activity, changes made in the C-terminal delivery unit retained at least some activity (Table 3). To further correlate the ability to kill certain bacteria and DnaK binding, we studied the interaction between the E. coli D-E helix DnaK fragment and fluorescein labeled Pyrr-mod1–4 peptides. In essence, the fluorescence anisotropy was not increased compared to the baseline when the inactive Pyrr-mod1 or Pyrr-mod2 peptides were added to the synthetic DnaK fragment. However, when the active Pyrr-mod3 and Pyrr- mod4 peptides were added, significant increases in the fluorescence anisotropy were detected, once again showing full correlation between DnaK binding and antimicrobial activity (Fig. 4). These experiments further confirmed the location of the pharmacophore (i.e. DnaK binding site) between Asp2 and Pro10 in pyrrhocoricin. It needs to be mentioned that in these labeled Pyrr-mod1–4 peptides, no cationic amino acid, needed for the antibacterial activity of N-terminally labeled pyrrhocoricin analogs [2], was inserted Table 3. In vitro antibacterial activity of modified pyrrhocoricin peptides. One quarter strength of Muller-Hinton broth was used as the medium for the growth of E. coli, A. tumefaciens, K. pneumoniae and P. aeruginosa; 1/4 strength of brain-heart infusion for S. typhimurium and H. influenzae (for the latter one the medium was supplemented with NAD) and poor broth medium was used for S. aureus. Peptide IC 50 (l M ) against E. coli A. tumefaciens S. typhimurium K. pneumoniae H. influenzae P. aeruginosa S. aureus Pyrrhocoricin < 0.1 < 0.3 2.5–5 1.25–2.5 2.5–5 10–20 > 20 Pyrr-mod1 > 20 > 40 > 20 > 20 > 20 > 20 > 20 Pyrr-mod2 > 20 > 40 > 20 > 20 > 20 > 20 > 20 Pyrr-mod3 < 0.1 0.6–1.25 5–10 5–10 5–10 > 20 > 20 Pyrr-mod4 0.1–-0.3 0.6–1.25 1.25–2.5 1.25–2.5 1.25–2.5 > 20 > 20 Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur. J. Biochem. 269) 4231 between the label and Val1. A combination of these data and those represented by Fig. 2 demonstrated that while an increased number of positive charges at the amino terminus was required for the gross antibacterial activity, the DnaK binding and cell distribution studies could proceed with analogs lacking the lysine insert. Conformation of the DnaK fragments Although we assumed that the lack of pyrrhocoricin binding to some of the DnaK D-E helix sequences directly reflected the lack of pyrrhocoricin activity against these bacteria, it was possible that alterations in the physical or biochemical properties of the synthetic protein fragments played roles in our inability to detect positive interaction between the peptides. One of the potential sources of altered behavior can be a change in the trademark helical conformation of the DnaK fragments. To exclude this scenario, we compared the secondary structure of the D-E helix peptides by CD spectroscopy. CD spectra were taken in water, and water/trifluoroethanol mixtures [30]. All DnaK fragments exhibited very similar spectra regardless of whether they bound to pyrrhocoricin or not. Here we present the data on E. coli and S. aureus (Fig. 5). In water, the CD spectra of both DnaK fragments could be assigned as a type C spectrum [31], and reflected the dominance of type I (III) b-turns, or a mixture of type I and type II turns [32]. Addition of 5% trifluoroethanol (v/v) resulted in a redshift of both pp* bands to 190 and 204 nm, respectively, accompanied by an increase of the intensity of the positive band which is a clear indication of the appearance of helical structures. The two DnaK fragments behaved identically, at least in spectral terms. While the spectral features of well-devel- oped a-helices could already be seen at a trifluoroethanol concentration as low as 10%, the intensity was increased with increasing trifluoroethanol content. The spectra of the E. coli and S. aureus peptides remained very similar in all water/trifluoroethanol compositions studied. If any difference could be detected it was a minor intensity increase throughout and some redshift (around 1 nm) at low trifluoroethanol concentrations for the S. aureus sequence compared to the E. coli peptide. This could be explained by the increased number of potential salt bridges along the helix barrel. The S. aureus peptide contained 3 and 5 potential Glu–Lys salt bridges in i,i+3 and i,i+4 positions, respectively. These figures for the E. coli fragment were 2 and 0. The isolated peptide fragments thus exhibited all helical features of the complete DnaK multihelical lid; the fragments were very similar but not identical. Having said this, pyrrhocoricin binding was not directly related to the helical content as determined by fluorescence polarization (data not shown). The millipolarization values between E. coli DnaK 583– 615 and Fl-K-pyrrhocoricin decreased as the trifluoroeth- anol content of the solvent increased in the 0–10% trifluoroethanol range. No fluorescence anisotropy could be detected when the solvent contained more the 10% trifluoroethanol. The CD studies further verified the binding of pyrrho- coricin to the E. coli DnaK fragment and the lack of a sequence analogous to that of S. aureus (Fig. 6). To study the interaction of the antibacterial peptide and the synthetic DnaK D-E helix fragments, we looked at the conformation of the E. coli DnaK 583–615 or the analogous S. aureus DnaK 554–585 peptide–pyrrhocoricin mixtures as shown by CD. If the sum of the individual CD curves of the two interacting partners is different from the CD of the ligand– receptor mixture, it is a clear indication of not only binding, but also of a conformational change upon interaction. Indeed, such a spectral alteration was detected for the binding fragment of the responsive strain E. coli, but not for the nonbinding fragment of the unresponsive strain S. aureus (Fig. 6). If any consequence for the nature of the Fig. 4. Interaction of 1 n M fluorescein-labeled pyrrhocoricin analogs with the E. coli DnaK 583–615 fragment. The concentration of the unlabeled protein fragment was 125 l M . The fluorescence anisotropy data are expressed as the difference in the millipolarization values between samples containing both the labeled antibacterial peptides and the E. coli receptor fragment, or the appropriate labeled pyrrhocoricin analogs alone. Fig. 3. Binding of fluorescein-labeled pyrrhocoricin to various synthetic DnaK D-E helix fragments as studied by fluorescence polarization. In these experiments, the N-terminally fluorescein-labeled pyrrhocoricin derivative at 1 n M concentration was added to synthetic D-E helix DnaK fragments that were applied at either 250 l M or 15 l M . The data are expressed as the difference in the millipolarization values between samples containing both the labeled antibacterial peptide and the given receptor fragments, or labeled pyrrhocoricin alone. The fig- ure shows the average of 10 readings taken after a 5 min incubation period. These experiments were repeated two times with basically identical outcomes. 4232 G. Kragol et al. (Eur. J. Biochem. 269) Ó FEBS 2002 conformational change was to be drawn, the decrease of the intensity of the 200 nm band representing the unordered structure suggests the generation of a more ordered conformation. Identification of the pyrrhocoricin binding surface on E. coli DnaK Encouraged by the success of the Ala-scan in identifying the essential residues of pyrrhororicin for antibacterial activity, we used a similar strategy to identify essential residues in E. coli DnaK for pyrrhocoricin binding. For this purpose each residue in the 583–610 D-E helix fragment was replaced with alanine. Phenylalanine was substituted for the three native alanines in the sequence. Binding to N-terminally fluorescein-labeled and Lys extended pyrrho- coricin was studied by fluorescence polarization. The fluorescence anisotropy showed a ladder-type interaction pattern that probably reflected the presence of vital residues for the binding and flanking residues likely needed to maintain the structural integrity at the interaction sites (Fig. 7). The major drop in the fluorescence polarization signal was observed when Glu7 (Glu589 in the full protein), Gln13 (Gln595) or Met16 (Met598) were replaced with Ala. Additional important residues were Gln6 (Gln588), Gln10 (Gln592), Leu15 (Leu597), Ala19 (Ala601), Gln20 (Gln602) and Gln22 (Gln604). Figure 7 shows an average of three independent measurements and is cut at 12 millipolarization units, which represents pyrrhocoricin binding to the native Fig. 6. Circular dichroism spectra of DnaK D-E helix–pyrrhocoricin mixtures in water. First, the individual peptide spectra were col- lected, followed by the spectra of the anti- bacterial peptide–protein fragment mixtures. While the measured spectrum of the E. coli fragment–pyrrhocoricin mixture is different from the mathematical sum of the individual spectra, and indicates a conformational change upon interaction, no similar alteration could be detected for the unresponsive strain S. aureus. Fig. 5. Circular dichroism spectra of E. coli and S. aureus DnaK D-E helix fragments in trifluoroethanol-water mixtures. The spectra were taken at room temperature and the peptide concentration was approximately 0.5 mgÆmL )1 . Both peptides underwent a characteristic turn–helix transition as the tri- fluoroethanol concentration increased. Fig. 7. Binding of fluorescein-labeled pyrrhocoricin to alanine-substi- tuted E. coli DnaK D-E helix analogs as studied by fluorescence polar- ization. In these experiments, the N-terminally fluorescein-labeled pyrrhocoricin derivative at 1 n M concentration was added to the Ala- scan of synthetic D-E helix DnaK fragments that were applied at 150 l M . The data are expressed as the difference in the millipolariza- tion values between samples containing both the labeled antibacterial peptide and the D-E helix analogs, or labeled pyrrhocoricin alone. The figure shows the average of three independent experiments conducted from freshly purified samples; in each, 10 readings were taken after a 5 min incubation period. Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur. J. Biochem. 269) 4233 E. coli DnaK 583–610 sequence. In reality, substitution of some residues increased rather than decreased the fluores- cence anisotropy. The reason for this finding is still unclear; it may indicate stronger binding to the mutated peptides, an increased fluorescence signal due to a somewhat shifted binding site or simply solubility differences. DISCUSSION Proline-rich delivery modules To reach the cell interior, antibacterial peptides have to cross the bacterial membrane. To fight pathogens living inside host cells, peptides generally have to enter eukaryotic cells as well. In the current study, we looked at the entry of pyrrhocoricin, drosocin and apidaecin into E. coli cells and macrophages, host cells of intracellular bacteria such as Mycobacterium tuberculosis or Mycobacterium leprae. The full-sized peptides entered the cells and became distributed in all cellular compartments, suggesting an efficient transport mechanism for these molecules. In our previous report, we suggested that in addition to the target protein, DnaK, the 60 kDa chaperonin protein family, GroEL in bacteria and Hsp60 in mammalian cells, can serve such a purpose [5]. Remarkably, when full-sized peptides were studied, no selective surface staining of the cells could be detected by confocal fluorescence microscopy. Such surface staining is characteristic for alternating arginine-leucine contain- ing biooligomers and other membrane-destabilizing antimicrobial peptides, including magainin 2 [18]. In con- trast, the PRP or similar repeat sequences in the proline-rich antibacterial peptide family assist the entry into the host and subsequently into bacterial cells without any potential to destabilize the cells, and therefore become toxic to eukar- yotes. The antibacterial activity of the native products is provided by the independently functioning active site, capable of binding to the D-E helix region of bacterial DnaK and preventing chaperone-assisted protein folding [6]. Proline-rich cell penetration modules may be general for antibacterial peptides in nature. Bovine myeloid cathelicidins carry precursors of broad spectrum antimicrobial peptides characterized by N-terminal amphipathic a-helices and C-terminal hydrophobic tails [33]. Antibacterial peptides with amphipathic helices or sheets are considered to act on the negatively charged bacterial membranes by creating a strong dipole moment and destroying the energy gradient across the membrane [34]. Indeed, the C-terminal tails of the cathelicidins are not needed for the antibacterial activity with the truncated peptides containing only the amphipathic helices being equally active against a selection of both Gram- negative and Gram-positive bacteria [33]. However, a 5- to 10-fold higher concentration of the truncated analogs is required to achieve a kinetics of permeabilization similar to that of the respective parent peptides, suggesting a somewhat less effective initial interaction with the target bacterial membranes [33]. An examination of the cathelicidin hydro- phobic tail sequences (PVIPLLHR and PIIVPIIRI) reveal strong similarities to C-terminal tails of pyrrhocoricin (PPRPIYNRN), drosocin (SHPRPIRV) or apidaecin (PHPRI). Apparently, regardless of the mechanism for peptide-mediated killing of bacteria, the highly similar cell- penetrating modules may serve as first interaction points with the membrane surface and help the internalization of the peptides. In turn, this may suggest that some individual antibacterial peptides or peptide families for which we originally assumed a membrane-only mode of action [1] may have intracellular targets too, just the sequences responsible for nonmembrane activity are disguised by the ubiquitous presence of membrane-targeting delivery modules. Interaction between pyrrhocoricin and bacterial biopolymers from the viewpoint of the antibacterial peptide The essential region of pyrrhocoricin for the antibacterial activity was located between Asp2 and Pro10. The identi- fication of the Asp2–Pro10 active segment is in full agreement with our earlier model of pyrrhocoricin–DnaK interactions. In our previous report [6], we showed a model of how pyrrhocoricin remains close to the E-helix of E. coli DnaK after applying a flexible docking algorithm. When we look at this model from the angle of the E helix barrel, it is evident that the pyrrhocoricin fragment that most intimately touches DnaK is exactly the experimentally identified Tyr6– Pro10 hexapeptide segment [35]. The pyrrhocoricin 1–9 fragment alone (or pyrrhocoricin 1–10 for that matter) penetrated into cells very inefficiently but was an absolute requirement for antibacterial activity. Pyrrhocoricin is shown to bind to DnaK both inside and outside the peptide-binding pocket [6]. The N-terminal region might play roles in the association with molecular chaperones (DnaK or GroEL) that serve as transport vehicles for the peptide. Consistent with this, an application of the predi- cative algorithm for nonspecific interaction of peptides with the conventional peptide binding pocket of E. coli DnaK [36] reveals a high probability of the N-terminal trideca- peptide fragment of pyrrhocoricin associating with the heat shock protein (energy score )5.0), but not when the 13-residue window starts from Gly4 (energy score +4.7). Significantly, the side-chain of Thr11, located at the border of the pharmacophore and the delivery unit, appeared not to drastically influence the antimicrobial activity. This observation is compatible with our earlier findings showing that the native sugar addition to Thr11 has no influence on the gross antibacterial activity [28]. The presence of the carbohydrate side-chain, however, may modify either the binding to the target protein or the ability of the peptide to penetrate cells via through-space effects. The importance of Arg19 was documented previously, when we reported that the deletion of the C-terminal Arg19–Asn20 dipeptide results in a reduction of the activity against A. tumefaciens [28]. The partial loss of activity after replacing the two C-terminal arginines suggests that this region is responsible for the initial interaction of pyrrhocoricin with the negat- ively charged bacterial surface and the ensuing entry into bacterial cells. The modified C-terminal delivery module featuring an increased number of the Arg–Pro segments had an advant- age against two strains that have more resistant membrane structures, S. typhimurium and H. influenzae. Even if the C-terminal fragment was not directly involved in binding to the target protein, it still interacted with a biopolymer in a stereospecific manner. The weak activity of native pyrrho- coricin against P. aeruginosa, a gram negative strain known to have an outer membrane that is difficult to penetrate [37], may reflect either only a low ability of the peptide to 4234 G. Kragol et al. (Eur. J. Biochem. 269) Ó FEBS 2002 disintegrate this membrane structure or very weak binding to the target protein. A Lys1-containing analog of pyrrho- coricin shows improved activity against both E. coli and P. aeruginosa, potentially shifting the dominant mode of action from DnaK inhibition to membrane disintegration [21]. N-Terminally blocked cecropin analogs, known to kill bacteria by disassembling the membrane structure, lack antibacterial activity [29]. All these suggest that the N-terminal charges play an equally important role in the interaction with bacterial membranes, but this might be decoupled from the distribution of the peptides in the cells. The N-terminally labeled Pyrr-mod4 analog without the extra lysine bound to DnaK, was well dispersed inside E. coli cells, but labeled a smaller number of bacterial or mammalian cells, and therefore was unable to kill E. coli (Table 2). It is tempting to speculate that antimicrobial peptides with intracellular targets form a loop and interact with the negatively charged bacterial membranes with both termini and not with only one side, as was impressively demonstrated previously [15]. Alternatively, the two charged domains could interact with spatially and tempor- ally separated bacterial components. Concerning the specific interaction between the antimi- crobial peptides and bacterial DnaK, the antibacterial activity and selective binding to the D-E helix region of the 70 kDa heat shock protein was strongly correlated. Pyr- rhocoricin bound to the representative synthetic fragments of the highly responsive strains E. coli, S. typhimurium, A. tumefaciens and H. influenzae,butfailedtobindtothe homologous fragments from the nonresponsive strains S. aureus, S. pyogenes, H. pylori, H. ducreyi, S. pneumoniae, C. albicans or the weakly responsive strain P. aeruginosa. Further support for the connection between binding to the D-E helix of DnaK and antibacterial activity came from the Pyrr-mod1–4 analogs. The antibacterial activity of pyrrho- coricin analogs that were modified in either the active site or in the C-terminal unit and binding to the protein fragment was strongly correlated. In addition, the native peptide lacked binding to the homologous region of mouse or human Hsp70 confirming the lack of specific peptide binding to full-sized human Hsp70 [5] or toxicity to mammalian cells and healthy mice [2]. Interaction between pyrrhocoricin and DnaK from the viewpoint of the heat shock protein To identify a potential pyrrhocoricin-binding surface on E. coli DnaK, here we analyzed the important residues for the interaction based on the published X-ray structure of the protein. In Fig. 8, we displayed the surface of the C-terminal domain (residues 533–606) in semitransparent gray [38], then marked the synthetic 583–606 fragment in ball and stick inside the surface. Due to the lack of ordered structure C-terminal to Ala606, the rest of the synthetic DnaK peptide is excluded from this graphical representation. The crucial residues for pyrrhocoricin binding based on Fig. 7 are colored in Fig. 8 blue (most important) and green (important). This exercise identifies an L-shaped binding surface, including the primary binder linear surface com- prising residues Gln13 (Gln595 in the full protein), Met16 (Met598), Ala19 (Ala601) and Gln20 (Gln602), and the secondary surface attached in an 80 degrees angle featuring Gln6 (Gln588) and Gln10 (Gln592). Additional essential residues, Lys14 (Lys596), Leu15 (Leu597) and Ile18 (Ile600) are involved in the stabilization of the correct fold of DnaK for pyrrhocoricin binding. Of these, Leu597 and Ile600 have no or very little surface exposure (calculated by [39]), but make hydrophobic contacts with neighboring essential residues. While Leu597 strongly influences the proper side-chain orientation of the highly important residue Met598, Ile600 touches the aliphatic portion of the side chain of Lys596. The apparently very significant residues Glu7 (Glu589) and Gln22 (Gln604) have surface exposure, but are facing the opposite direction. These hydrophilic residues probably maintain the aqueous solubility required for the successful fluorescence polarization assay of the otherwise not very soluble synthetic DnaK D-E helix peptide fragment. This model would well explain the lack of activity against all the negative bacterial strains and higher organisms. H. pylori, P. aeruginosa, S. aureus, S. pyogenes, S. pneumo- niae, C. albicans, M. musculus and H. sapiens all lack both Met16 and Gln20 (compare with Table 1). These residues are present in the responsive strain H. influenzae and the homologous unresponsive strain H. ducreyi. The two Fig. 8. Possible pyrrhocoricin-interacting resi- dues in E. coli DnaK. The published structure of the C-terminal domain of E. coli DnaK [38] is displayed in semitransparent. The synthetic 583–606 fragment, used for the Ala-scan is marked with ball and stick representation inside the surface. The crucial residues for pyrrhocoricin binding based on the Ala-scan are labeled blue (most important) and green (important). The likely primary and secondary binding surfaces are highlighted by blue and green patches, respectively. Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur. J. Biochem. 269) 4235 [...]... Structure -activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cellpenetrating ability of buforin II Proc Natl Acad Sci USA 97, 8245–8250 Oren, Z., Hong, J & Shai, Y (1997) A repertoire of novel antibacterial diastereomeric peptides with selective cytolytic activity J Biol Chem 272, 14643–14649 Hancock, R.E.W & Diamond, J (2000) The role of. .. and another fully homologous stretch of residues until Gln606 followed by nine nonidentical residues (Table 1) By coincidence, in E coli residue 601 was found important for binding, but because of the altered residues in H infleunzae and H ducreyi in positions 596 and 600, a valine in H influenzae might allow stronger binding than an alanine in H ducreyi Alternatively, the residues responsible for the positive... Otvos, L Jr (1999) Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects Biochim Biophys Acta 1426, 459–467 29 Vunnam, S., Juvvadi, P & Merrifield, R.B (1997) Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from the pig intestine J Pept Res 49, 59–66 30 Nelson, J.W & Kallenbach, N.R (1986) Stabilization of the ribonuclease S-peptide... characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities J Biol Chem 271, 28375–28381 34 Gudmundsson, G.H & Agerbeth, B (1999) Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system J Immunol Methods 232, 45–54 35 Otvos, L (2002) The short, proline-rich antibacterial. .. the residues responsible for the positive pyrrhocoricin binding in H influenzae DnaK and negative binding in H ducreyi DnaK are located outside the 533–606 fragment used for modeling or 583–610 fragment used for the mutational analysis It is possible that the pyrrhocoricin binding site may be shifted in other bacteria compared to E coli If we look at the sequence of A tumefaciens this is indeed a viable... a glutamine are found in positions 13 and 17, exactly three residues N-terminal to the crucial Met16 (Met598) and Gln20 (Gln602) of E coli DnaK Multiple peptide-binding sites on heat shock proteins were most recently shown to exist, both in thermodynamic and kinetic experiments [40,41] The precise assignment of the contact residues between pyrrhocoricin and E coli, H influenzae and A tumefaciens DnaK... Hoffmann, R & Otvos, L Jr (2001) The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding Biochemistry 40, 3016–3026 7 Wu, M., Maier, E., Benz, R & Hancock, R.E.W (1999) Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli Biochemistry... scale chemical synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide from Drosophila Eur J Biochem 238, 64–69 Ó FEBS 2002 Multifunctional antimicrobial peptides (Eur J Biochem 269) 4237 27 Szendrei, G.I., Fabian, H., Mantsch, H.H., Lovas, S., Nyeki, O., Schon, I & Otvos, L Jr (1994) Aspartate-bond isomerization affects the major conformations of synthetic peptides Eur... Otvos, L Jr (2001) The efficacy of the antibacterial peptide, pyrrhocoricin, is finely regulated by its amino acid residues and active domains Lett Pept Sci 8, 201–209 Fields, G.B & Noble, R.L (1990) Solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl amino acids Int J Pept Protein Res 35, 161–214 Lundblad, J.R., Laurence, M & Goodman, R.H (1996) Fluorescence polarization analysis of protein-DNA... J.D & Hoffmann, R (1998) Glycosylation of the fourth repeat unit of human s-protein abolishes binding to the C-terminal acidic s-binding segment of b-tubulin Prot Pept Lett 5, 207–213 Hoffmann, R., Craik, D.J., Pierens, G., Bolger, R.E & Otvos, L Jr (1998) Phosphorylation of the C-terminal sites of human p53 reduces non-sequence specific DNA binding as modeled with synthetic peptides Biochemistry 37, 13755–13764 . Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin Goran Kragol 1 , Ralf Hoffmann 2 ,. 110. RESULTS Identification of crucial residues for the antibacterial activity of pyrrhocoricin An Ala-scan was performed to identify key residues that could