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• W2089348575 abstract "The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C. The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 °C. The bacterial membrane became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys18 and Arg4) are distributed into three clusters on the surface of hRNase7. The first cluster, K1,K3,K111,K112, was located at the flexible coil near the N terminus, whereas the other two, K32,K35 and K96,R97,K100, were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, and His123 or other clusters such as K32,K35 and K96,R97,K100, is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K1,K3,K111,K112. The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 °C. Ribonucleases (RNases) 3The abbreviations used are: RNase, ribonuclease; hRNase, human RNase; cfu, colony-forming unit(s); CD, circular dichroism; HSQC, heteronuclear single quantum correlation; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. are found widely among living organisms, and they play an important role in the metabolism of RNA (1.D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Recently, novel biological functions other than the intrinsic ribonucleolytic activity have been demonstrated for several members of the bovine RNaseA superfamily. In the human RNase family, eosinophil-derived neurotoxin (RNase2) and eosinophil cationic protein (RNase3) have neurotoxic (2.Durack D.T. Ackerman S.J. Loegering D.A. Gleich G.J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5165-5169Crossref PubMed Scopus (185) Google Scholar) as well as antiparasitic activity (3.McLaren D.J. McKean J.R. Olsson I. Venges P. Kay A.B. Parasite Immunol. 1981; 3: 359-373Crossref PubMed Scopus (101) Google Scholar). Angiogenin (RNase5), which induces blood vessel formation, is also an RNase (4.Fett J.W. Strydom D.J. Lobb R.R. Alderman E.M. Bethune J.L. Riordan J.F. Vallee B.L. Biochemistry. 1985; 24: 5480-5486Crossref PubMed Scopus (845) Google Scholar). In the frog RNase family, most of the members have antitumor activity (5.Liao Y.D. Huang H.C. Leu Y.J. Wei C.W. Tang P.C. Wang S.C. Nucleic Acids Res. 2000; 28: 4097-4104Crossref PubMed Scopus (41) Google Scholar). For the above-mentioned functions, the catalytic activities of RNases are essential. Some members of RNase superfamily, such as human RNases 3, 5, and 7 (hRNase3, hRNase5, and hRNase7), mouse angiogenins 1 and 4 (mAng1 and mAng4), as well as chicken RNaseA2, also have antimicrobial activities (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar, 7.Harder J. Schroder J.M. J. Biol. Chem. 2002; 277: 46779-46784Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 8.Hooper L.V. Stappenbeck T.S. Hong C.V. Gordon J.I. Nat. Immunol. 2003; 4: 269-273Crossref PubMed Scopus (739) Google Scholar, 9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar, 10.Nitto T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The mechanisms for these antibacterial activities remain unclear, although some distinct properties exist in these bactericidal RNases. First of all, the calculated isoelectric points of bactericidal RNases are higher than those of non-bactericidal RNases. Second, the net positive charges of the bactericidal RNases are higher than those of non-bactericidal RNases (Table 1). There is no evidence, however, that the high positive charge or high pI value is critical for the antimicrobial activity. In this study, we have presented evidence that one flexible Lys cluster K1,K3,K111,K112, rather than the catalytic residues His15, Lys38, His123 or other Lys clusters such as K32,K35, and K96,R97,K100,is critical for the bactericidal activity of hRNase7. The functional role of these Lys residues (K1,K3,K111,K112) on the bactericidal activity of hRNase7 will be discussed.TABLE 1Properties of selected RNases in bovine RNase A superfamilyRNaseFunctionCatalytic activityAmino acid no.LysArgAspGluNet chargespIdAccession codes are as follows: hRNase 2 (X16546), hRNase 3 (X16545), hRNase 7 (NM_032572), hRNase 5 (NM_001145), mAng (NM_007447), mAng-4 (AY219870), bRNase A (X07283), RC-RNase (AF039104), and RC-RNase 6 (AF242556)hRNase 2Antiviral, neurotoxic0.65aZhang, et al. (6)1344841+79.20hRNase 3Antimicrobial0.048aZhang, et al. (6)13311960+1410.72hRNase 7Antibacterial0.021aZhang, et al. (6)12818442+169.80hRNase 5Angiogenesis, antibacterialExtremely lowa,Zhang, et al. (6)bHolloway, et al. (9)12371364+109.73mAngAngiogenesis, antibacterial30% of hRNase 5bHolloway, et al. (9)12191174+99.54mAng-4AntibacterialUnknownbHolloway, et al. (9)120111077+79.20RNase ARNA catabolism1085.6cLiao, et al. (5)12410455+48.64RC-RNaseAntitumor964.4cLiao, et al. (5)1114612+79.20RC-RNase6Antitumor0.007cLiao, et al. (5)10515582+109.39a Zhang, et al. (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar)b Holloway, et al. (9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar)c Liao, et al. (5.Liao Y.D. Huang H.C. Leu Y.J. Wei C.W. Tang P.C. Wang S.C. Nucleic Acids Res. 2000; 28: 4097-4104Crossref PubMed Scopus (41) Google Scholar)d Accession codes are as follows: hRNase 2 (X16546), hRNase 3 (X16545), hRNase 7 (NM_032572), hRNase 5 (NM_001145), mAng (NM_007447), mAng-4 (AY219870), bRNase A (X07283), RC-RNase (AF039104), and RC-RNase 6 (AF242556) Open table in a new tab Preparation and Enzymatic Assay of RNases−The coding region of the human RNase7 (hRNase7) gene was obtained from the genomic DNA of HeLa cells by PCR. The NdeI and BamHI restriction sites were hung on the 5′ and 3′ end of the gene fragment, respectively, and subcloned into the T7 RNA polymerase-driven expression vector pET22b (Novagen) (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). Site-directed mutagenesis was made by PCR as previously described (12.Huang H.C. Wang S.C. Leu Y.J. Lu S.C. Liao Y.D. J. Biol. Chem. 1998; 273: 6395-6401Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The uniformly 15N- or 13C-labeled hRNase7 with an extra Met at the N terminus was produced in Escherichia coli BL21(DE3). The recombinant hRNase7 from inclusion bodies were refolded and purified as previously described (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). The ribonucleolytic activity of recombinant hRNase7 was analyzed by the zymogram assay on RNA-casting PAGE as previously described (13.Liao Y.D. Wang J.J. Eur. J. Biochem. 1994; 222: 215-220Crossref PubMed Scopus (30) Google Scholar). Antimicrobial Activity Assay−The bacteria were cultured in Nutrient broth and plated on Nutrient agar (Difco 0001) for Pseudomonas aeruginosa Migula (American Type Culture Collection (ATCC) 27853), tryptic soy broth/agar (Difco 0369) for Staphylococcus aureus subspecies Aureus Rosenbach (ATCC 6538P), and Luria-Bertani broth/agar for E. coli DH5α. The yeast Candida albicans (Robin) Berkhout (ATCC 14053) was cultured and plated in/on yeast malt broth/agar, and Pichia pastoris X-33 was cultured and plated in/on yeast extract-peptone-dextrose broth/agar. The microbes were grown overnight, washed, and diluted 1:100 in 10 mm sodium phosphate, pH 7.4. 45 μl of the microbes (5–10 × 104 colony-forming units (cfu)) was mixed with various concentrations of RNase/oligopeptide (5 μl), which was dissolved in 20 mm Hepes, pH 7.4, 50 mm NaCl, and incubated at 37 °C for 3 h. Serial dilution of each RNase-treated bacteria/yeast was prepared and plated for the determination of the remaining cfu (14.Rosenberg H.F. Dyer K.D. J. Biol. Chem. 1995; 270 (Correction (1995) J. Biol. Chem.270, 30234): 21539-21544Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Binding of RNase to Bacteria−Small aliquots of RNases (2 μg in 5 μl) were incubated with 45 μl(5 × 106 cfu) of sodium phosphate-washed P. aeruginosa at 37 °C for 30 min. The RNases bound onto bacteria were spun down, washed five times with 10 mm sodium phosphate, and analyzed by SDS-PAGE and silver staining. Assays of the Permeability of Bacterial Membrane−The overnight culture of P. aeruginosa (107 cfu) was washed and resuspended in 100 μl of water and incubated with 1 μm SYTOX® Green (Molecular Probes) in a dark 96-well plate for 15 min in the dark. After the addition of RNase, the increase of fluorescence due to the binding of the dye to the intracellular DNA was measured in the same microplate reader using 485- and 520-nm filters for excitation and emission wavelengths, respectively (15.Nekhotiaeva N. Elmquist A. Rajarao G.K. Hallbrink M. Langel U. Good L. FASEB J. 2004; 18: 394-396Crossref PubMed Scopus (122) Google Scholar). Nomenclature of Mutated hRNase7 and Oligopeptide−Some of the hRNase7 mutants are designated as follows: K1A (substitution of Lys1 by Ala); H15A (substitution of His15 by Ala); K32N/K35Q (substitution of Lys32 by Asn and Lys35 by Gln); K96A/R97A/K100T (substitution of Lys96 by Ala, Arg97 by Ala, and Lys100 by Thr); K111Q/K112Q (substitution of Lys111 by Gln and Lys112 by Gln); Δ1KPKG4 (deletion of N-terminal Met and Lys1-Pro2-Lys3-Gly4 residues from the recombinant hRNase7; and (R3S1)3, (12-mer oligopeptide containing the triple-repeated Arg-Arg-Arg-Ser sequence). Circular Dichroism (CD) Experiments−CD experiments were carried out using an Aviv CD 202 spectrometer (Lakewood, NJ) using a 1-mm path length cuvette with 20 μm hRNase7 in 20 mm sodium phosphate. The CD spectra at different temperatures and pH values were recorded from 190 to 260 nm using a wavelength step of 0.5 nm. Equilibrium thermal denaturing experiments were performed using protein samples dissolved in 20 mm phosphate buffer, pH 3.5, by measuring the change of molar ellipticity at 201 nm. Data were collected as a function of temperature with a scan rate of 2 °C/min and allowing 3 min to reach equilibrium over the range of 4–95 °C. Equilibrium unfolding induced by guanidine HCl was monitored by CD as described previously (11.Hsu C.H. Liao Y.D. Pan Y.R. Chen L.W. Wu S.H. Leu Y.J. Chen C. J. Mol. Biol. 2003; 326: 1189-1201Crossref PubMed Scopus (17) Google Scholar). The curves were fitted and analyzed using SigmaPlot version 8.02 (SPSS Inc.). NMR Spectroscopy−All NMR experiments were performed on Bruker AVANCE 600 and 800 spectrometers equipped with triple (1H, 13C, and 15N) resonance probes, including a shielded z-gradient. The RNase samples (0.6 mm in 0.35 ml) were prepared in 50 mm phosphate buffer in 90% H2O/10% D2Oor 99.9% D2O at pH 3.5, 310 K and kept in a Shigemi NMR tube. All heteronuclear NMR experiments were carried out as described previously (16.Clore G.M. Gronenborn A.M. Methods Enzymol. 1994; 239: 349-363Crossref PubMed Scopus (253) Google Scholar). Sequence-specific assignment of the backbone atoms of hRNase7 was achieved by the independent connectivity analysis of CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, and C(CO)NH. The 1H resonances were assigned using TOCSY-HSQC, HAHB(CO)NH, and HCCH-TOCSY. Combined information from two-dimensional 1H-15N HSQC and three-dimensional NOESY-HSQC experiments yielded assignments for side chain amide resonances of the Asn and Gln residues. Aromatic resonances were assigned using two-dimensional 1H-13C HSQC, NOESY, and TOCSY data. Linear prediction was used in the 13C and 15N dimensions to improve the digital resolution. All spectra were processed using the NMRPipe software package (17.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar) and analyzed using NMRView version 5.0 (18.Johnson B.A. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar). NMR Restraints and Tertiary Structure Calculation of hRNase7−The dihedral angle information was predicted by the TALOS program (19.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). The hydrogen bonding information was obtained from D2O exchange monitored by two-dimensional 1H-15N HSQC spectra. The nuclear Overhauser effect restraints from 1H-15N NOESY-HSQC and 1H-13C NOESYHSQC spectra were assigned by the automated CANDID module of CYANA (20.Herrmann T. Guntert P. Wuthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1329) Google Scholar) and then checked manually. NMR structures were calculated based on all experimental restraints by simulated annealing using the program Xplor-NIH. The final 15 structures that contained the lowest total energies, no distance restraint violation >0.3 Å, and no dihedral angle restraint violation >3 ° were chosen. The distributions of the backbone dihedral angles of the final converged structures were evaluated by the representation of the Ramachandran dihedral pattern, which shows the deviations from the sterically allowed (ϕ, Ψ) angle limits, by using PROCHECK-NMR (21.Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4429) Google Scholar) and MOLMOL software (22.Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (29-32): 51-55Crossref PubMed Scopus (6490) Google Scholar). Data Bank Accession Number−The chemical shifts of recombinant hRNase7 at pH 3.5 and 310 K have been deposited in the BioMagResBank under accession number BMRB-7206. The best 15 structures, together with the complete list of restraints, have been deposited in the Brookhaven Protein Data Bank under accession number 2HKY. Antimicrobial Activity of RNase Superfamily Proteins−The hRNase7 was more effective (0.1 μm for 102-fold reduction) in cfu compared with that of buffer only against bacteria (105 cfu P. aeruginosa) than bullfrog RC-RNase6 (5 μm), whereas the active RNA-degrading RNases (bovine RNaseA and bull-frog RC-RNase) and all other bullfrog oocytic RNases were not bactericidal at 80 μm. The 12-mer Arg-rich positively charged oligopeptide (R3S1)3, however, possessed similar bactericidal activity (0.2 μm) as that of hRNase7 (0.1 μm) (Fig. 1, A and B). The antimicrobial spectrum of hRNase7 was also examined, and the yeast P. pastoris X-33 (0.03 μm for 102-fold reduction in cfu compared with that of buffer only) was the most sensitive among all of the microbes tested and thereafter in order the Gram-negative bacterium P. aeruginosa (0.1 μm), whereas the Gram-positive S. aureus (1 μm) and Gram-negative E. coli bacteria (10 μm) and yeast C. albicans (> 20 μm) were not sensitive (Fig. 1, B and C). Binding and Permeability to Bacterial Membrane−The cfu of susceptible bacteria was reduced 102-fold in only a few minutes after RNase addition (data not shown). The bactericidal RNases (hRNase7 and RC-RNase6) were bound to the susceptible bacterium P. aeruginosa (Fig. 2, lanes 2 and 11). In contrast, the non-bactericidal bovine RNaseA and bullfrog RC-RNase did not bind to the bacteria (Fig. 2, lanes 5 and 8). The membrane of P. aeruginosa became permeable to the DNA-binding dye SYTOX® Green in only a few minutes after the addition of 2.5 μm hRNase7 or bullfrog RC-RNase6 to the bacteria (107 cfu in 100 μl). However, the non-bactericidal bovine RNaseA and bullfrog RC-RNase had no effect under the same conditions, which was in good agreement with the antimicrobial assay (Figs. 1A and 3). The bactericidal activity of hRNase7 was only slightly reduced at 4 °C (0.3 μm for 102-fold reduction in cfu compared with that of 0.1 μm at 37 °C), whereas that of indolicidin, a 13-mer bactericidal oligopeptide from bovine neutroplils, was almost abolished (Fig. 4). These results suggest that the bactericidal activity of hRNase7 is not energy-dependent, whereas that of indolicidin is energy-dependent.FIGURE 4The effect of temperature on the bactericidal activity of hRNase7 and indolicidin. The antimicrobial activities of hRNase7 and indolicidin against P. aeruginosa at both 37 °C (A) and 4 °C (B) were determined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conformational and Structural Stability of hRNase7−To elucidate the mechanism for the antimicrobial activity of hRNase7, the structure of the recombinant protein was analyzed by CD spectra and NMR studies. The important features are summarized and discussed below. First, the conformation of hRNase7 was pH-independent over the range of 3.5–9.5. The Tm value of hRNase7 was 66.4 °C. The chemical stability was determined with a Cm value at 3.27 m guanidine-HCl. These Tm and Cm values indicate that hRNase7 is a stable protein in structure, similar to other RNaseA superfamily members. Structure of hRNase7−The 800-MHz two-dimensional 1H-15N HSQC spectrum of the hRNase7 was obtained in which cross-peaks clearly dispersed (Fig. 5). For the determination of tertiary structures, a set of 1661 restraints were collected for simulated annealing calculations. Among these restraints, 1447 were interproton distances, 94 were hydrogen bonds, 116 were torsional angles, and 4 were disulfide bond restraints (Table 2). The 15 structures of the lowest total energy were chosen to represent the ensemble of NMR structures (Fig. 6A). These structures were consistent with both experimental data and standard covalent geometry and displayed no violations >0.3 Å for distance restraints and no violations >3 ° for torsional angles. Superposition of each structure with the mean structure yielded an average root mean square deviation of 0.34 ± 0.09 Å for the backbone atoms and 1.06 ± 0.08 Å for the heavy atoms in residues 7–128. Analysis of the ensemble using PROCHECK-NMR revealed that 64.4% of the residues lay in the most favored regions and 33.9% of the residues lay in allowed regions in the Ramachandran ϕ, Ψ dihedral-angle plot (Table 2).TABLE 2Structural statistics on the final set of 15 simulated annealing structures for human RNase 7Constraints usedNOE distance restraintsIntraresidue (|i–j| = 0)211Sequential (|i–j| = 1)363Medium range (1<|i–j| < 5)335Long range (|i–j| ≥ 5)538Total NOE distance restraints1447Hydrogen bonds47 × 2Disulfide bonds4Dihedral angles116Statistics for the final X-PLOR structuresNumber of structures in the final set15X-PLOR energy (kcal · mol–1)ENOE131.3 ± 4.0Ecdih3.3 ± 0.4Ebond + Eangle + Eimproper231.1 ± 6.0EVDW322.0 ± 6.4Mean global root mean square deviation (Å)Backbone atoms (N, Cα, C′) (residues 7–128)0.34 ± 0.09Backbone atoms (N, Cα, C′) (secondary structure)0.24 ± 0.06Heavy atoms (residues 7–128)1.06 ± 0.08Heavy atoms (secondary structure)0.95 ± 0.10Ramachandran plot (%)Residues in most favored regions64.4Residues in allowed regions26.9Residues in generously allowed regions7.0Residues in disallowed regions1.7 Open table in a new tab FIGURE 6Structure of hRNase7. A, NMR solution structures of hRNase7. Left, the ensemble of 15 NMR solution structures is displayed. The α-helical structures are shown in red, β-strands in blue, and other secondary structures are colored in gray. Right, the ribbon representation of the best NMR structure that possesses the lowest total energy is shown. The hRNase7 is composed of three α-helices (red) and two triple-stranded antiparallel β-sheets (cyan). B, surface structure of hRNase7 is displayed as a 180 ° rotation with positively and negatively charged residues shown in blue and red, respectively. C, side chain conformations of all charged residues, Lys (K) in blue, Arg (R) in cyan, Asp (D) in red, and Glu (E) in pink, of 15 NMR structures of hRNase7 are displayed as a 180 ° rotation. The figure was generated based on the superposition of the backbone atoms in the full-length protein, and clarification of only one ribbon structure is shown. The charged residues that were mutated for antimicrobial activity studies are boxed with dotted lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The solution structure of hRNase7 displayed an α + β folding topology, which was composed of three α-helices and two antiparallel β-sheets, typical for the RNaseA superfamily. The structure was stabilized by four disulfide bridges (C23–C81, C55–C106, C37–C91 and C62–C69). Most of the hydrogen bonds were located in the α-helix and β-sheet regions. Most positively charged residues were distributed into three clusters (Fig. 6B). The first was composed of Lys1, Lys3, Lys111, and Lys112; the second, Lys28, Lys32, Lys35, Arg36, and Lys38, and the third, Lys82, Lys94, Lys96, Arg97, and Lys100. On the other hand, the negatively charged residues were randomly distributed over hRNase7 (Fig. 6C). Residues Responsible for Bactericidal Activity of hRNase7−Because cationic residues were abundant in bactericidal RNases as demonstrated in Table 1, we thus proceeded to determine the role of the RNA catalytic activity or cationic residue in the bactericidal activity. First of all, we found that the catalytic activity-deficient mutants of hRNase7 (H15A, K38A, and H123A) still conferred the same level of antimicrobial activity as the wild-type hRNase7 (Fig. 7, A and B; and Fig. 8A). The result shows that RNA catalytic activity is not essential for the antibacterial activity of RNases.FIGURE 8Antimicrobial activities of recombinant hRNase7 and mutants. A, catalytic RNA-deficient mutants. B and C, deletion or substitution mutants of positively charged residues. Δ1KPKG4 represents the deletion of N-terminal Met and 1KPKG4 residues from the recombinant hRNase7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Deletion of the N-terminal Met and four residues (Δ1KPKG4) from recombinant hRNase7 markedly reduced the bactericidal activity but did not alter the RNA catalytic activities (Fig. 7, C and D; and Fig. 8B). Further analysis showed that the K3A mutant exerted less bactericidal activity than the K1A mutant (Fig. 7, C and D; and Fig. 8C). However, the substitution of the K111Q/K112Q residues in the same cationic cluster reduced the bactericidal activity to a lesser extent than N-terminal deletion. In contrast, mutations in other positively charged clusters (K32N/K35Q and K96A/R97A/K100T) altered neither the catalytic nor the bactericidal activity (Fig. 7, C and D; and Fig. 8B). These results show that the flexible Lys1,Lys3, as well as Lys111,Lys112 residues in the first cationic cluster are critical for the bactericidal activity. Structural Comparison of Wild-type and K3A Mutated hRNase7− The structure of K3A-hRNase7 was nearly identical to that of wild-type hRNase7, as there was no difference in the cross-peaks of two-dimensional N15-H1-HSQC and three-dimensional N15-NOESY and HSQC NMR spectra between them, except residues Gly4, Leu124, and Ala3, although K3A-hRNase7 had less antimicrobial activity than wild-type hRNase7 (Fig. 8C). The structures of bactericidal RNases hRNase3 and hRNase7 are similar to those of RNaseA superfamily proteins with three α-helices and two triple-stranded antiparallel β-sheets. Among these RNases, only the bactericidal RNases contained abundant positively charged residues on the enzyme surface, but the non-bactericidal RNases did not (Table 1). The abundance of the cationic residues Lys and Arg is also found in most antimicrobial peptides. These peptides possess amphipathic structures that are composed of clustered cationic and hydrophobic residues on each side of the structure, although they have diverse primary sequences and different secondary structures (23.Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1151) Google Scholar, 24.Hancock R.E. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar). The cationic residues may facilitate their interaction with the negatively charged components on the microbial surface, and the hydrophobic residues may permit their incorporation into microbial membrane (25.Powers J.P. Hancock R.E. Peptides. 2003; 24: 1681-1691Crossref PubMed Scopus (742) Google Scholar). In this study, we have found that three clusters of cationic residues are located on the surface of hRNase7. The first cluster consists of Lys1,Lys3, Lys111,Lys112 residues, the second and third clusters contain Lys32,Lys35 and Lys96,Arg97,Lys100, respectively. Only cationic residues in the first cluster, K1,K3,K111,K112, are critical for the bactericidal activity, whereas the other two clusters, K32,K35, and K96,R97,K100, are not (Figs. 6A and 8B). The result of Fig. 6C shows that the side chains of the Lys1, Lys3, Lys111, and Lys112 residues in the first cluster are more flexible than those of cationic residues in other clusters. In addition, we also found that no anionic residue resides in the first cluster of hRNase7 near the K1,K3,K111,K112 residues by NMR studies (Fig. 6) and no anionic residue resides at each N-terminal region of bactericidal RNases by the comparison of amino acid sequences among the RNaseA superfamily members (Fig. 9). Thus, it is concluded that clustering of cationic residues without an adjacent anionic residue is critical for the bactericidal activity of hRNase. Several members in the RNase superfamily have antimicrobial activities, but the key residues/domains responsible for antimicrobial activity were different in human RNase3 and RNase7 (6.Zhang J. Dyer K.D. Rosenberg H.F. Nucleic Acids Res. 2003; 31: 602-607Crossref PubMed Scopus (86) Google Scholar, 7.Harder J. Schroder J.M. J. Biol. Chem. 2002; 277: 46779-46784Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 8.Hooper L.V. Stappenbeck T.S. Hong C.V. Gordon J.I. Nat. Immunol. 2003; 4: 269-273Crossref PubMed Scopus (739) Google Scholar, 9.Holloway D.E. Hares M.C. Shapiro R. Subramanian V. Acharya K.R. Protein Expression Purif. 2001; 22: 307-317Crossref PubMed Scopus (39) Google Scholar, 10.Nitto T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The content of cationic residues of hRNase3 (Lys1 and Arg19) differs from that of hRNase7 (Lys18 and Arg4). Furthermore, the cationic residues Arg101 and Arg104 and aromatic residues Trp10 and Trp35 of hRNase3, which are suggested to be responsible for membrane binding and disruption, reside on the dispersed secondary structure (α1, β4) (26.Carreras E. Boix E. Rosenberg H.F. Cuchillo C.M. Nogues M.V. Biochemistry. 2003; 42: 6636-6644Crossref PubMed Scopus (87) Google Scholar), whereas the cationic residues K1,K3,K111,K112, of hRNase7 reside on the flexible coil and loop at the N-terminal cluster. With regard to chicken RNaseA2, the Arg residues in domains II (residues 71–76) and III (residues 89–104) are critical for the bactericidal activity, but the structures of the RNaseA2 remains unknown (10.Nitto T. Dyer K.D. Czapiga M. Rosenberg H.F. J. Biol. Chem. 2006; 281: 25622-25634Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition to the flexibility of the side chains of cationic residues, we were also interested in the influence of similar tertiary structure folds for RNases on the antimicrobial activity. Our results indicate that the bactericidal activity of RNase was not correlated with its backbone tertiary structure; for example, the root mean square deviation values between hRNase7 and bactericidal hRNase3 and hRNase5 are 2.57 and 3.37 Å, respectively, whereas those between bactericidal hRNase7 and non-bactericidal hRNase2, hRNase4, and bovine RNaseA are 2.34, 3.09, and 2.33 Å, respectively. This indicates that the tertiary structure fold of the whole RNase backbone is not critical for the antimicrobial activity. The hRNase7 was effective in bactericidal activity at 4 °C, whereas the indolicidin, a Trp-rich oligopeptide (ILPWKWWPWWPWRR-NH2) from bovine neutrophils, was not (27.Hsu C.H. Chen C. Jou M.L. Lee A.Y. Lin Y.C. Yu Y.P. Huang W.T. Wu S.H. Nucleic Acids Res. 2005; 33: 4053-4064Crossref PubMed Scopus (232) Google Scholar). The bactericidal RNases hRNase7 and RC-RNase6 from bullfrog oocytes were bound to susceptible bacteria, whereas the non-bactericidal RNases were not. These results suggest that some component(s) on the bacteria is/are responsible for the binding of hRNase7 and the action mechanism of hRNase7 is energy-independent (28.Falla T.J. Karunaratne D.N. Hancock R.E. J. Biol. Chem. 1996; 271: 19298-19303Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 29.Robinson Jr., W.E. McDougall B. Tran D. Selsted M.E. J. Leukocyte Biol. 1998; 63: 94-100Crossref PubMed Scopus (174) Google Scholar). The bacterial membrane became permeable to the DNA binding dye SYTOX® Green just a few minutes after the addition of hRNase7 (Fig. 3). This indicates that the marked increase of membrane permeability is an important step for the bactericidal activity of hRNase7. Thus, we suggest that the hRNase7 may bind to the negatively charged components of the bacterial membrane through the flexible and cationic residues. The hRNase7 may change its own conformation, incorporates itself into the bacterial membrane through the hydrophobic scaffold, and triggers the disruption of membrane. Alternatively, we also propose that several pores, which are composed of hRNase7-binding proteins, may reside on the bacterial surface for the regulation of the transportation of ions and metabolites. The opening of pores may be triggered by hRNase7 binding through the flexible/clustered cationic residues and hydrophobic scaffold. The efflux of ions and fatal depolarization of bacterial membrane may thus cause immediate cell death, even at low temperatures. The antibacterial mechanisms of these positively charged peptides/proteins by physical disruption of bacterial membrane is different from those for conventional antibiotics, which are inhibition of cell wall synthesis, DNA replication, RNA transcription, or protein synthesis. Due to the unique bactericidal activity of hRNase7, it has the potential to be a new therapeutic agent for bacterial infection, because it may not face the rapid emergence of drug resistance. The induction of endogenous hRNase7 gene expression or the administration of a synthetic oligopeptide designed from the hRNase7 structure would be possible in the clinical therapy for microbial infection. We thank Dr. Chen-Pei David Tu for critical reading of the manuscript, Dr. Yuan-Chao Lou for editing the manuscript, and Chiu-Feng Wang for the construction of various hRNase7 mutants." @default.
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• W2089348575 title "The Flexible and Clustered Lysine Residues of Human Ribonuclease 7 Are Critical for Membrane Permeability and Antimicrobial Activity" @default.
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