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• W2040941372 abstract "Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM. Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM. The Gram-positive bacterium Streptococcus pyogenes (group A streptococcus, GAS) 3The abbreviations used are: GAS, group A streptococcus; PAM, plasminogen binding group A streptococcal M-like protein.3The abbreviations used are: GAS, group A streptococcus; PAM, plasminogen binding group A streptococcal M-like protein. is responsible for a wide variety of skin and mucosal infections in humans. Current estimates indicate that ∼1.78 million new cases of severe streptococcal infection occur each year (1Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. Lancet. Infect. Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1880) Google Scholar). A key feature of invasive GAS infections is the ability of the organism to migrate from cutaneous and mucosal surfaces to deep tissue sites, resulting in severe invasive disease. The binding and activation of plasminogen by GAS has been implicated in the pathogenesis of this organism (2Walker M.J. McArthur J.D. McKay F. Ranson M. Trends Microbiol. 2005; 13: 308-313Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Plasminogen is a single chain glycoprotein found in plasma and extracellular fluids at concentrations of ∼2 μm (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2289) Google Scholar). Cleavage of plasminogen at a single site (Arg560-Val561) by specific plasminogen activators results in the formation of the two-chain plasmin molecule, which contains a serine protease active site in the C-terminal region (4Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar). Human plasminogen can also be activated to plasmin by the GAS protein streptokinase, as part of a highly species-specific plasminogen/streptokinase activator complex (2Walker M.J. McArthur J.D. McKay F. Ranson M. Trends Microbiol. 2005; 13: 308-313Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Plasmin has the ability to degrade fibrin clots, connective tissue, and the extracellular matrix (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2289) Google Scholar, 4Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar). Thus activation of this proteolytic system by GAS may have significant pathological consequences in the host (5Sun H. Ringdahl U. Homeister J.W. Fay W.P. Engleberg N.C. Yang A.Y. Rozek L.S. Wang X. Sjobring U. Ginsburg D. Science. 2004; 305: 1283-1286Crossref PubMed Scopus (313) Google Scholar). Isolated initially from M53 serotype GAS, the plasminogen-binding group A streptococcal M-like protein (PAM) is a 42-kDa molecule that binds both plasmin and plasminogen directly and with high affinity (Kd ∼ 1 nm) (6Berge A. Sjobring U. J. Biol. Chem. 1993; 268: 25417-25424Abstract Full Text PDF PubMed Google Scholar). A newly developed model of GAS infection using mice expressing a human plasminogen transgene indicates that plasminogen plays a critical role in GAS infection, with a significant increase in mortality observed in transgenic mice when compared with wild-type littermate control mice (5Sun H. Ringdahl U. Homeister J.W. Fay W.P. Engleberg N.C. Yang A.Y. Rozek L.S. Wang X. Sjobring U. Ginsburg D. Science. 2004; 305: 1283-1286Crossref PubMed Scopus (313) Google Scholar). Additionally, the PAM-positive GAS isolate AP53 exhibited a 60% increase in mortality in transgenic mice when compared with wild-type littermates, and a PAM-negative isogenic mutant of this strain showed only minimal virulence in both wild-type and human plasminogen transgenic murine backgrounds (5Sun H. Ringdahl U. Homeister J.W. Fay W.P. Engleberg N.C. Yang A.Y. Rozek L.S. Wang X. Sjobring U. Ginsburg D. Science. 2004; 305: 1283-1286Crossref PubMed Scopus (313) Google Scholar). Thus, it appears that for a subset of GAS isolates, the ability of PAM to focus plasminogen at the GAS cell surface is crucial for virulence. The major plasmin(ogen)-binding site of PAM is located in the N-terminal variable region of the protein and is composed of two characteristic tandem repeats designated a1 and a2. Similar binding motifs have been identified in M-like proteins of other GAS isolates associated with skin infection (6Berge A. Sjobring U. J. Biol. Chem. 1993; 268: 25417-25424Abstract Full Text PDF PubMed Google Scholar, 7Svensson M.D. Sjobring U. Bessen D.E. Infect. Immun. 1999; 67: 3915-3920Crossref PubMed Google Scholar). PAM has been implicated in the establishment of GAS skin infections such as impetigo, and the PAM genotype is almost exclusively associated with the chromosomal emm pattern D, which is considered a genetic marker for skin tropic GAS (7Svensson M.D. Sjobring U. Bessen D.E. Infect. Immun. 1999; 67: 3915-3920Crossref PubMed Google Scholar, 8Svensson M.D. Sjobring U. Luo F. Bessen D.E. Microbiology. 2002; 148: 3933-3945Crossref PubMed Scopus (61) Google Scholar, 9Bessen D.E. Sotir C.M. Readdy T.L. Hollingshead S.K. J. Infect Dis. 1996; 173: 896-900Crossref PubMed Scopus (138) Google Scholar). However, in the Northern Territory of Australia, where streptococcal skin infection is endemic and high rates of invasive infection such as bacteremia have been reported (10Carapetis J.R. Walker A.M. Hibble M. Sriprakash K.S. Currie B.J. Epidemiol. Infect. 1999; 122: 59-65Crossref PubMed Scopus (76) Google Scholar), PAM-positive GAS are associated with a variety of disease states (11McKay F.C. McArthur J.D. Sanderson-Smith M.L. Gardam S. Currie B.J. Sriprakash K.S. Fagan P.K. Towers R.J. Batzloff M.R. Chhatwal G.S. Ranson M. Walker M.J. Infect. Immun. 2004; 72: 364-370Crossref PubMed Scopus (67) Google Scholar). The plasminogen binding domain of PAM has been shown to be highly variable; however, this diversity does not abrogate plasminogen binding by naturally occurring PAM variants (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This finding may, in part, be due to the conservation of key amino acid residues within the plasminogen binding domain (11McKay F.C. McArthur J.D. Sanderson-Smith M.L. Gardam S. Currie B.J. Sriprakash K.S. Fagan P.K. Towers R.J. Batzloff M.R. Chhatwal G.S. Ranson M. Walker M.J. Infect. Immun. 2004; 72: 364-370Crossref PubMed Scopus (67) Google Scholar, 12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Plasmin-(ogen) is known to interact with its ligands via pre-formed lysine-binding sites within the N-terminally located triple-disulfide-bonded kringle domains (4Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar). Kringles 1, 4, and 5 display the highest affinity for lysine-based ligands, with kringle 2 displaying the weakest affinity (13Marti D.N. Hu C.K. An S.S. von Haller P. Schaller J. Llinas M. Biochemistry. 1997; 36: 11591-11604Crossref PubMed Scopus (60) Google Scholar). PAM lacks the typical C-terminal lysine residues characteristic of many plasminogen receptors. Rather, internal lysine residues in the a1 and a2 repeat regions of PAM (Lys98 and Lys111) are thought to mediate binding to kringle 2 of plasminogen (14Wistedt A.C. Ringdahl U. Muller-Esterl W. Sjobring U. Mol. Microbiol. 1995; 18: 569-578Crossref PubMed Scopus (84) Google Scholar). Early reports suggested that Lys98 of the a1 repeat contributed the majority of the plasminogen binding ability of PAM (14Wistedt A.C. Ringdahl U. Muller-Esterl W. Sjobring U. Mol. Microbiol. 1995; 18: 569-578Crossref PubMed Scopus (84) Google Scholar). However, studies involving a polypeptide designated VEK-30 and a recombinant kringle 2 modified to contain a high affinity lysine binding site, highlighted a potential role for internal His102, Arg101, and Glu104 residues within the a1 repeat in this interaction (15Schenone M.M. Warder S.E. Martin J.A. Prorok M. Castellino F.J. J. Pept. Res. 2000; 56: 438-445Crossref PubMed Scopus (22) Google Scholar, 16Rios-Steiner J.L. Schenone M. Mochalkin I. Tulinsky A. Castellino F.J. J. Mol. Biol. 2001; 308: 705-719Crossref PubMed Scopus (55) Google Scholar). VEK-30 is composed of the six residues preceding the a1 repeat, the entire a1 repeat, together with the first ten residues of the a2 repeat of PAM (17Wistedt A.C. Kotarsky H. Marti D. Ringdahl U. Castellino F.J. Schaller J. Sjobring U. J. Biol. Chem. 1998; 273: 24420-24424Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The relative contribution to binding by residues within the a1 and a2 repeat of the full-length PAM protein to plasminogen has not been characterized. Here, we have undertaken site-directed mutagenesis studies on the PAM molecule and defined residues involved in the interaction with plasminogen. These studies highlight the important contribution of multiple arginine and histidine residues within both the a1 and a2 plasminogen binding domains and may explain the capacity of naturally occurring PAM variants to maintain high affinity plasminogen binding despite considerable variation observed in the plasminogen binding repeats. Bacterial Strains and Culture Methods—Escherichia coli TOP10 containing pGEX2T-PAMNS13 expression plasmids were grown on Luria Bertani (LB) agar plates or cultured in LB broth supplemented with ampicillin (100 μg/ml) as described previously (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was extracted for PCR and DNA sequence analysis using the Wizard® Plus SV DNA purification kit (Promega). Site-directed Mutagenesis—The PAM gene from streptococcal strain NS13 (PAMNS13 GenBank™ AY351851), which is identical to the prototype PAM sequence in the a1 and a2 repeat domain, had previously been cloned into the expression vector pGEX2T (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To assess the role of specific-binding site residues in the interaction of PAM with plasminogen, site-specific mutations were introduced into the PAMNS13-binding site. The wild-type construct (100 ng) was used as template DNA to create site-directed mutants with the QuikChange™ site-directed mutagenesis kit (Stratagene). PCR reactions consisted of 1× Pfu reaction buffer (Stratagene), 0.25 mm dNTPs (Roche Applied Science), 2.5 units of Pfu Ultra polymerase (Stratagene), and 125 ng of each primer, made up to a volume of 50 μl with dH2O. Oligonucleotide primers (Sigma-Aldrich) were designed as per the manufacturer's instructions (Stratagene). In general, primers consisted of sequence encoding the desired mutation, flanked on either side by fifteen nucleotides of wild-type sequence. The specific primer sequences are given in Table 1, with the introduced mutations underlined. For site-directed mutants containing more than two mutations not encoded by a single primer, alanine residues were sequentially introduced using previously mutated DNA as a template. Following an initial denaturation step (95 °C for 30 s), PCR cycling parameters consisted of 16 cycles of 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 10 min. An additional 7-min extension at 55 °C was then performed. PCR was conducted with a Cooled Palm 96 thermocycler (Corbett Research). Non-amplified template DNA was removed by incubation of reactions for 1 h at 37 °C following the addition of DpnI (10 units/50-μl reaction). E. coli TOP10 (Invitrogen) were transformed with 150 ng of DpnI-digested PCR product using standard procedures (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar).TABLE 1Forward primers used for PCR construction and DNA sequence analysis of PAMNS13 site-directed mutantsIntroduced mutationForward mutagenesis primerLys98/Ala5′-GATGCTGAGTTGCAACGACTTGCAAACGAGAGACATGAAGAAGCA-3′Lys111/Ala5′-GAAGCAGAGTTGGAGCGACTTGCAAGCGAGAGACATGATCATGAC-3′Arg101-His102-Glu104/Ala5′-CGACTTGCAAACGAGGCAGCAGAAGCAGCAGAGTTGGAGCGA-3′Arg101-His102/Ala5′-CGACTTGCAAACGAGGCAGCAGAAGCAGCAGAGTTGGAGCGA-3′Arg114-His115/Ala5′-CGACTTGCAAGCGAGGCAGCAGATCATGACAAAAAAGAAGC-3′PrimerDNA sequence analysis primersPAMF15′-ATAAGCAAGAACATCTTGACGG-3′PAMR15′-CTGTTAATTTCTTGCTTTC-3′PAMF25′-AAAGGGCTTAAGACTGATTTAC-3′PAMR25′-GACCAGCTAATTTGCTGTTTGC-3′PAMF35′-GCAAACAGCAAATTAGCTGCTC-3′PAMR35′-CTTCTCAACATCATCTTTAAGG-3′pGEX2TF5′-GGGCTGGCAAGCCACGTTTGGTG-3′pGEX2TR5′-CCGGGAGCTGCATGTGTCAGAGG-3′ Open table in a new tab DNA Sequence Analysis, Expression, and Purification of Recombinant M Proteins—DNA sequence analysis was used to verify introduced mutations and confirm the absence of random mutations in site-directed mutants. DNA sequence analysis was performed using the primers listed in Table 1, and sequence reactions were undertaken using terminator ready reaction mix (PE Applied Biosystems). DNA sequencing gels were prepared as per the manufacturer's instructions and electrophoresed using an Applied Biosystems 3130 X genetic analyzer (Applied Biosystems). Sequence data were analyzed using ABI Prism™ DNA sequencing analysis software (PerkinElmer Life Sciences). Recombinant proteins were expressed and purified essentially as described previously (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar), from 1 liter of E. coli cultures, using glutathione-agarose (Sigma-Aldrich) and native nickel-nitrilotriacetic acid-agarose (Qiagen, Germany) affinity chromatography. Prior to elution from the glutathione-agarose column, the glutathione S-transferase tag was removed from the N terminus of recombinant fusion proteins by the addition of one column volume of thrombin solution (1 unit of thrombin/μl, phosphate-buffered saline, pH 8.0). The column was incubated for 5 h at room temperature, and the cleaved recombinant protein was eluted in phosphate-buffered saline (pH 8.0). Each step of the protein purification process was monitored by 12% SDS-polyacrylamide gel electrophoresis (PAGE) analysis (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar), with protein visualized using Coomassie R-250 staining. Circular Dichroism Spectroscopy—To examine potential variation in protein secondary structure as a result of site-directed mutagenesis, far-UV CD spectra were obtained for both wild-type and mutant recombinant PAM proteins. CD spectra were acquired using a J-810 spectropolarimeter (Jasco, Victoria, British Columbia, Canada) at room temperature. CD spectra data were recorded from 190 to 250 nm in a 1-cm path length cell containing 1.5 ml of protein solution at a concentration of 0.04 mg/ml in 10 mm sodium phosphate buffer (pH 7.4). Recorded data represent the average of six scans, corrected for buffer baseline. Molar residue ellipticity ([θ]) was calculated using the following formula: [θ] = θ × 100 × molecular weight/concentration (mg/ml) × distance × number of amino acids (21Schmid F.X. Creighton T.E. Protein Structure: A Practical Approach. IRL Press at Oxford University Press, Oxford, UK1989: 251-284Google Scholar). The percentage of α-helix was estimated from the ellipticity at 222 nm using the following formula: % α-helix = –(θ222 nm – 4,800)/45,400 (22Phillips Jr., G.N. Flicker P.F. Cohen C. Manjula B.N. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4689-4693Crossref PubMed Scopus (195) Google Scholar). Plasminogen Purification and Labeling—Glu-plasminogen was purified from human plasma using lysine Sepharose-4B affinity chromatography as described previously (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Andronicos N.M. Ranson M. Bognacki J. Baker M.S. Biochim. Biophys. Acta. 1997; 1337: 27-39Crossref PubMed Scopus (50) Google Scholar). Purified plasminogen was biotinylated by the addition of 10% (v/v) 1 m NaHCO3 (pH 9), and a 40 m excess of biotin-X-N-hydroxy-succinimide in Me2SO (Sigma-Aldrich). The reaction was incubated at 4 °C overnight with mixing. Free biotin was separated from biotinylated plasminogen by PD-10 gel-filtration chromatography (Amersham Biosciences) (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Plasminogen Binding Analysis—Solid-phase plasminogen binding assays were performed to assess the impact of introduced mutations on protein function essentially as previously described (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Ninety-six well microtiter plates (Greiner Bioone, Germany) were coated with 150 nm recombinant protein (50 μl in 0.1 m NaHCO3) at 4 °C overnight. Following three washes with PiNT (50 mm Na2HPO4, 150 mm NaCl, 0.05% Tween 80, pH 7.5), plates were blocked with 50 μl of blocking solution (1% skim milk powder, PiNT) for 1 h at 37°C. Wells were washed as above, and 500 nm biotinylated Glu-plasminogen was diluted in a 3-fold titration across the plate with blocking buffer, in the presence or absence of a 50-fold molar excess of unlabeled Glu-plasminogen. Plasminogen was allowed to bind to immobilized proteins for 2 h at room temperature. For competition assays, decreasing concentrations of unlabeled fluid-phase wild-type PAMNS13 (25 μm to 0.14 nm) were allowed to compete with immobilized proteins for binding to biotinylated Glu-plasminogen. Competitor was titrated 3-fold across the microtiter plate prior to the addition of biotinylated Glu-plasminogen to all wells, at a final concentration of 500 nm. The assay was incubated for 2 h at room temperature. Following the plasminogen incubation step, microtiter plates were washed three times, 50 μl of NeutrAvidin conjugated to horseradish peroxidase (Progen, Australia) diluted 1:5000 with blocking solution was added to all wells, and the mixture was incubated for 2 h at room temperature. After five washes with PiNT, the reactions were developed by the addition of 50 μl of o-phenylenediamine (Sigma-Aldrich) substrate (8 mm Na2HPO4, pH 5.0, 2.2 mm o-phenylenediamine, 3% H2O2). Color development was stopped by the addition of 50 μl of 10 m hydrochloric acid, and the plates were read at 490 nm using a Spectramax 250 plate reader (Molecular Devices). Data were normalized against the highest and lowest absorbance value for each assay, and non-linear regression analysis was performed using GraphPad® Prism (version 4.00, GraphPad software, San Diego, CA). For the calculation of equilibrium binding dissociation constants (Kd), a one- versus two-site binding analysis was conducted, and the best-fit curve was fitted to the data. For competition experiments, a one-site competition curve was fitted to the data from which the effective concentration of competitor required to inhibit binding by 50% (EC50) was calculated. Statistical Analysis—For plasminogen binding experiments, a one-way analysis of variance was initially used on all data, followed by an unpaired t test with Welsch's correction to determine if there was any significant difference in the Kd values for plasminogen binding by PAM mutants and PAMNS13. The gene encoding wild-type PAMNS13 shares 100% identity with the prototype PAM sequence in the a1 and a2 repeat domain responsible for the interaction with plasminogen (11McKay F.C. McArthur J.D. Sanderson-Smith M.L. Gardam S. Currie B.J. Sriprakash K.S. Fagan P.K. Towers R.J. Batzloff M.R. Chhatwal G.S. Ranson M. Walker M.J. Infect. Immun. 2004; 72: 364-370Crossref PubMed Scopus (67) Google Scholar). To assess the role of specific binding site residues in the interaction of PAMNS13 with plasminogen, a number of site-directed mutants were constructed in which the residues of interest were replaced with alanine. The binding site sequences of the five constructed mutants are shown in Fig. 1A. The presence of introduced mutations was verified by DNA sequence analysis. Following expression in E. coli, recombinant proteins of ∼40 kDa in size were purified using glutathione-agarose and nickel-nitrilotriacetic acid-agarose affinity chromatography (Fig. 1B). The impact of mutations in the a1 and a2 repeat domains of PAMNS13 on protein structure were analyzed using far-UV CD spectroscopy. All proteins used in this study were found to have a CD emission spectrum characteristic of α-helical coiled proteins (24Freifelder D.M. Physical Biochemistry. Applications to Biochemistry and Molecular Biology. Second. W.H. Freeman and Co., New York1982Google Scholar), displaying two characteristic minima at ∼210 and 220 nm and a maximum peak at 190 nm (Fig. 2). This is similar to the CD spectra of other streptococcal M proteins, which are coiled-coil α-helical proteins (22Phillips Jr., G.N. Flicker P.F. Cohen C. Manjula B.N. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4689-4693Crossref PubMed Scopus (195) Google Scholar). Thus, even after mutagenesis these proteins appear to maintain an α-helical secondary structure. Additionally, for all proteins the two minima are of a similar magnitude, which is indicative of coiled-coil proteins (25Graddis T.J. Myszka D.G. Chaiken I.M. Biochemistry. 1993; 32: 12664-12671Crossref PubMed Scopus (147) Google Scholar). Percent α-helicity ranged from 27% to 44% (Table 2). CD analysis of other streptococcal M proteins has found them to contain between 23% and 70% α-helix (22Phillips Jr., G.N. Flicker P.F. Cohen C. Manjula B.N. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4689-4693Crossref PubMed Scopus (195) Google Scholar, 26Khandke K.M. Fairwell T. Acharya A.S. Trus B.L. Manjula B.N. J. Biol. Chem. 1988; 263: 5075-5082Abstract Full Text PDF PubMed Google Scholar).TABLE 2Functional and structural characteristics of PAMNS13 site-directed mutantsProteinKdEC50α-Helixnmμm%PAMNS131.5822.0627PAMNS13[Lys98-Lys11]10.340.5741PAMNS13[Lys98-Arg101-His102-Glu104-Lys111]50.240.2533PAMNS13[Lys98-Arg101-His102-Glu104-Lys111-Arg114-His115]Non-specific binding onlyNDaND, not determined.44PAMNS13[Arg101-His102]1.694.8733PAMNS13[Arg101-His102-Arg114-His115]Non-specific binding onlyND29a ND, not determined. Open table in a new tab PAMNS13 has previously been shown to interact with Glu-plasminogen with high affinity (Kd ∼ 1 nm) (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To determine the impact of the introduced mutations in the PAMNS13 binding site on plasminogen binding, solid-phase binding assays were performed utilizing biotinylated Glu-plasminogen. The recombinant PAM mutants bound plasminogen in a dose-dependant fashion, and saturable binding was achieved with 500 nm plasminogen for three of the five mutant proteins after 2 h (Fig. 3). Non-linear regression analysis was used to determine the affinity of each recombinant protein for Gluplasminogen (Table 2). Equilibrium dissociation constants (Kd) were calculated using a best-fit non-linear regression curve. The following site-directed mutants where residues were replaced with alanine, PAMNS13[Lys98–Lys111] and PAMNS13[Lys98–Arg101–His102–Glu104–Lys111], bound plasminogen with Kd values of 10.34 and 50.24 nm, respectively. Although this represents a significant decrease in affinity for Glu-plasminogen when compared with wild-type PAMNS13 (Kd = 1.58 nm; p < 0.05), binding by these mutants was still specific and saturable. PAMNS13[Arg101–His102] bound plasminogen with a Kd value of 1.69 nm, which is not significantly different fromthat of wild-type PAMNS13 (p > 0.05). Only nonspecific plasminogen binding was seen for mutants PAMNS13[Lys98–Arg101–His102–Glu104–Lys111–Arg114–His115] and PAMNS13[Arg101–His102–Arg114–His115], indicating that the arginine and histidine residues in both repeat domains are critical residues in the interaction of PAM with plasminogen. To further explore the contribution of these residues to the interaction with plasminogen, competition binding experiments were performed. The effective concentration of competitor required to inhibit plasminogen binding by 50% (EC50) was determined by fitting a one-site competition curve (Fig. 4). EC50 values ranged from 0.25 to 22.06 μm. This represents a >90% decrease in EC50 values for mutant proteins PAMNS13[Lys98–Lys111], PAMNS13[Lys98–Arg101–His102–Glu104–Lys111], and PAMNS13[Arg101–His102], when compared with wild-type PAMNS13. As expected, there was an inverse correlation between Kd and EC50 (Table 2). These data indicate that, although mutation of residues Lys98, Arg101, His102, Glu104, and Lys111 within the plasminogen binding repeats of PAMNS13 decreases the avidity of the interaction with plasminogen, the simultaneous mutation of residues Arg101, His102, Arg114, and His115 is required to fully abolish binding. A key feature of certain strains of S. pyogenes is the ability to migrate from cutaneous and mucosal surfaces to deep tissue sites, resulting in serious invasive infections such as bacteremia, necrotizing fasciitis, and streptococcal toxic shock-like syndrome. The exact mechanisms of GAS-invasive infection have yet to be fully explained, however one hypothesis involves the interaction of GAS with the host plasminogen activation system. Four GAS plasminogen-binding proteins have been described in the literature, as well as the secreted plasminogen activator streptokinase (2Walker M.J. McArthur J.D. McKay F. Ranson M. Trends Microbiol. 2005; 13: 308-313Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27Lahteenmaki K. Kuusela P. Korhonen T.K. FEMS Microbiol. Rev. 2001; 25: 531-552Crossref PubMed Scopus (275) Google Scholar, 28Sanderson-Smith M.L. McKay F.C. Ranson M. Walker M.J. Curr. Trends Microbiol. 2004; 1: 75-85Google Scholar, 29Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1731) Google Scholar). The multiplicity of potential virulence factors associated with S. pyogenes that interact with the plasminogen activation system necessitates a deeper understanding of the relationship of GAS with plasminogen. PAM is a cell surface-exposed, high affinity plasminogen receptor expressed by GAS associated with a variety of disease states, and it appears to play an integral role in the plasminogen-dependent virulence of PAM-positive GAS. In a recent study, it was shown that elimination of PAM-dependent plasminogen binding by GAS significantly reduced mortality in mice expressing the human plasminogen transgene (5Sun H. Ringdahl U. Homeister J.W. Fay W.P. Engleberg N.C. Yang A.Y. Rozek L.S. Wang X. Sjobring U. Ginsburg D. Science. 2004; 305: 1283-1286Crossref PubMed Scopus (313) Google Scholar). Furthermore, although the plasminogen binding domain of PAM is highly variable, this variation does not appear to significantly impact on the high affinity interaction of PAM with plasminogen. The conservation of binding function despite sequence diversity is indicative of the physiological significance of this interaction, and the ability of GAS to subvert the host plasminogen activation system (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The a1 and a2 repeat domains in the N terminus of PAM mediate binding to kringle 2 of plasminogen, with the a1 repeat residue Lys98 generally considered to make the greatest contribution to this interaction (6Berge A. Sjobring U. J. Biol. Chem. 1993; 268: 25417-25424Abstract Full Text PDF PubMed Google Scholar, 14Wistedt A.C. Ringdahl U. Muller-Esterl W. Sjobring U. Mol. Microbiol. 1995; 18: 569-578Crossref PubMed Scopus (84) Google Scholar, 17Wistedt A.C. Kotarsky H. Marti D. Ringdahl U. Castellino F.J. Schaller J. Sjobring U. J. Biol. Chem. 1998; 273: 24420-24424Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Using site-directed mutagenesis, this study indicates that residues Arg101 and His102 within the a1 repeat, together with residues Arg114 and His115 within the a2 repeat, are critical for the interaction of full-length PAM with plasminogen. The interaction with plasminogen of site-directed mutant PAMNS13 [Lys98–Lys111] in which residues Lys98 and Lys111 were mutated to alanines was dose-dependant and specific. Additionally, the Kd value for this interaction was within a physiologically relevant concentration of plasminogen, which circulates in the bloodstream at a concentration of ∼2 μm (3Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2289) Google Scholar). Furthermore, mutation of binding site residues Arg101, His102, Arg114, and His115 abolished plasminogen binding by PAMNS13 despite the presence of residues Lys98 and Lys111. To our knowledge, this is the first demonstration of a non-lysine-dependent, high affinity interaction between plasminogen and a full-length naturally occurring receptor. Previous studies involving the interaction of a polypeptide sequence designated VEK-30 with modified kringle 2 of plasminogen highlighted the importance of PAM a1/a2 residues Lys98, Arg101, His102, Glu104, and Lys111 in the peptide-plasminogen interaction (15Schenone M.M. Warder S.E. Martin J.A. Prorok M. Castellino F.J. J. Pept. Res. 2000; 56: 438-445Crossref PubMed Scopus (22) Google Scholar, 16Rios-Steiner J.L. Schenone M. Mochalkin I. Tulinsky A. Castellino F.J. J. Mol. Biol. 2001; 308: 705-719Crossref PubMed Scopus (55) Google Scholar). Notably, VEK-30 lacks the final three residues of the a2 repeat (Arg114, His115, and Glu116). Mutation of residues Lys98, Arg101, His102, Glu104, and Lys111 in the full-length protein PAMNS13 in this study did not eliminate plasminogen binding. However, the avidity of this interaction was significantly reduced, as evidenced by a >90% decrease in EC50 values recorded for mutant proteins when compared with wild-type PAMNS13. This supports the previous finding that these residues are important in the interaction of PAM with plasminogen. However, the finding that mutation of arginine (Arg101 and Arg114) and histidine (His102 and His115) residues in both the a1 and a2 repeat was required to fully abrogate plasminogen binding suggests that both repeats are able to mediate high affinity interactions with plasminogen. Residues Arg101 and His102 of the a1 repeat of PAM have been shown to make numerous salt bridge and hydrophobic electrostatic interactions with recombinant kringle 2, forming a pseudo-ligand similar to the lysine analogue ϵ-aminocaproic acid (16Rios-Steiner J.L. Schenone M. Mochalkin I. Tulinsky A. Castellino F.J. J. Mol. Biol. 2001; 308: 705-719Crossref PubMed Scopus (55) Google Scholar). It is thus likely that the corresponding residues Arg114 and His115 in the a2 repeat interact with plasminogen in a similar fashion. The decrease in plasminogen binding by site-directed mutants reported here does not appear to be due to loss of secondary structure, because all site-directed mutants displayed CD spectra characteristic of coiled-coil α-helical proteins, similar to that of the wild-type PAMNS13. The percent α-helicity for mutants reported here was between 27% and 44%. CD analysis of other streptococcal M proteins has found them to contain between 23% and 70% α-helix (22Phillips Jr., G.N. Flicker P.F. Cohen C. Manjula B.N. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4689-4693Crossref PubMed Scopus (195) Google Scholar, 26Khandke K.M. Fairwell T. Acharya A.S. Trus B.L. Manjula B.N. J. Biol. Chem. 1988; 263: 5075-5082Abstract Full Text PDF PubMed Google Scholar). However, the spectra for all α-helical proteins are not identical due to the small effect of non-aromatic side chains on the rotary strength of the peptide bond and occasional helix distortions (24Freifelder D.M. Physical Biochemistry. Applications to Biochemistry and Molecular Biology. Second. W.H. Freeman and Co., New York1982Google Scholar). It has also been shown that the α-helical structure of the peptide VEK-30, representative of the a1 repeat of PAM, increases from 25% to 75% upon binding to kringle 2 of plasminogen (16Rios-Steiner J.L. Schenone M. Mochalkin I. Tulinsky A. Castellino F.J. J. Mol. Biol. 2001; 308: 705-719Crossref PubMed Scopus (55) Google Scholar). It is possible that a similar structural shift could occur in the full-length PAM protein. No correlation was seen between percent α-helicity and plasminogen binding function, suggesting that any secondary structure changes have had only a minor influence on the differences in plasminogen binding described here. We have recently shown that, although the plasminogen binding domain of PAM is highly variable, this diversity does not abrogate plasminogen binding by naturally occurring PAM variants (12Sanderson-Smith M. Batzloff M. Sriprakash K.S. Dowton M. Ranson M. Walker M.J. J. Biol. Chem. 2006; 281: 3217-3226Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In these naturally occurring PAM variants, the Arg and His residues in both a1 and a2 repeat domains are highly conserved (11McKay F.C. McArthur J.D. Sanderson-Smith M.L. Gardam S. Currie B.J. Sriprakash K.S. Fagan P.K. Towers R.J. Batzloff M.R. Chhatwal G.S. Ranson M. Walker M.J. Infect. Immun. 2004; 72: 364-370Crossref PubMed Scopus (67) Google Scholar). Interestingly, in this study, a loss of plasminogen binding was only observed following simultaneous mutation of both the Arg101 and His102 residues in the a1 repeat, and the Arg114 and His115 residues in the a2 repeat. Therefore, conservation of Arg and His residues in either repeat domain may compensate for variation elsewhere in the binding repeats and explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM. Recent findings that the acquisition of plasminogen by S. pyogenes may be crucial for the virulence of certain strains of GAS, and the ability of multiple GAS proteins to facilitate this process, necessitates a deeper understanding of the mechanisms via which certain GAS proteins interact with plasminogen. This study highlights for the first time the importance of highly conserved arginine (Arg101 and Arg114) and histidine (His102 and His115) residues within the binding site of the plasminogen-binding protein PAM, in mediating the interaction of this molecule with plasminogen. Such a finding may have implications for the identification of novel plasminogen-binding proteins in the future. We thank Dr. Teresa Treweek for her assistance with the CD analysis." @default.
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