Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252 + 255A, SENK304A, SENK334A, SENK344E, SENK435L, and SENΔ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SENK344E had lost α-enolase activity, which was also reduced in SENK334A and SENΔ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SENK252 + 255A, SENK435L, and SENΔ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures. The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252 + 255A, SENK304A, SENK334A, SENK344E, SENK435L, and SENΔ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SENK344E had lost α-enolase activity, which was also reduced in SENK334A and SENΔ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SENK252 + 255A, SENK435L, and SENΔ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures. Streptococcus pyogenes (group A Streptococcus, GAS) 8The abbreviations used are: GASgroup A StreptococcusSENsurface enolaseMSmass spectrometryIMion mobilityESIelectrospray mass ionizationToftime-of-flightmbarmillibar. 8The abbreviations used are: GASgroup A StreptococcusSENsurface enolaseMSmass spectrometryIMion mobilityESIelectrospray mass ionizationToftime-of-flightmbarmillibar. is a common bacterial pathogen, causing over 700 million human disease episodes 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 (1882) Google Scholar). 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Andreasen P.A. Grøndahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2289) Google Scholar). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg561–Val562 site (23Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20Parry M.A. Zhang X.C. Bode I. Trends Biochem. Sci. 2000; 25: 53-59Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23Ponting C.P. Marshall J.M. Cederholm-Williams S.A. Blood Coagul. Fibrinolysis. 1992; 3: 605-614Crossref PubMed Scopus (199) Google Scholar). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20Parry M.A. Zhang X.C. Bode I. Trends Biochem. Sci. 2000; 25: 53-59Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 24Reddy K.N. Markus G. J. Biol. Chem. 1972; 247: 1683-1691Abstract Full Text PDF PubMed Google Scholar). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α2-antiplasmin and α2-macroglobulin (11Kuusela P. Ullberg M. Saksela O. Kronvall G. Infect. Immun. 1992; 60: 196-201Crossref PubMed Google Scholar, 20Parry M.A. Zhang X.C. Bode I. Trends Biochem. Sci. 2000; 25: 53-59Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6Walker 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). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25Berge A. Sjöbring U. J. Biol. Chem. 1993; 268: 25417-25424Abstract Full Text PDF PubMed Google Scholar), the PAM-related protein (19Sanderson-Smith M.L. Dinkla K. Cole J.N. Cork A.J. Maamary P.G. McArthur J.D. Chhatwal G.S. Walker M.J. FASEB J. 2008; 22: 2715-2722Crossref PubMed Scopus (62) Google Scholar), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (505) Google Scholar, 26Lottenberg R. Broder C.C. Boyle M.D. Kain S.J. Schroeder B.L. Curtiss 3rd, R. J. Bacteriol. 1992; 174: 5204-5210Crossref PubMed Google Scholar), and streptococcal surface enolase (SEN or α-enolase) (27Pancholi V. Fischetti V.A. J. Biol. Chem. 1998; 273: 14503-14515Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6Walker 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). In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27Pancholi V. Fischetti V.A. J. Biol. Chem. 1998; 273: 14503-14515Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 28Pancholi V. Cell. Mol. Life Sci. 2001; 58: 902-920Crossref PubMed Scopus (710) Google Scholar, 29Wold F. Boyer P.D. The Enzymes, Vol. 5. 3rd Ed. Academic Press, New York1971: 499-538Google Scholar). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27Pancholi V. Fischetti V.A. J. Biol. Chem. 1998; 273: 14503-14515Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 28Pancholi V. Cell. Mol. Life Sci. 2001; 58: 902-920Crossref PubMed Scopus (710) Google Scholar, 30Cole J.N. Ramirez R.D. Currie B.J. Cordwell S.J. Djordjevic S.P. Walker M.J. Infect. Immun. 2005; 73: 3137-3146Crossref PubMed Scopus (91) Google Scholar, 31Bergmann S. Hammerschmidt S. Thromb. Haemost. 2007; 98: 512-520Crossref PubMed Scopus (146) Google Scholar). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27Pancholi V. Fischetti V.A. J. Biol. 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D. on for Scholar). this of we a of for octameric SEN of the A of SEN at and a primarily structure of the that the structure which with the of the in silico functional analysis of SEN to the enzymatic activity and ability to bind human The α-enolase activity of SEN of 2-phosphoglycerate to was at The of phosphoenolpyruvate for SEN was of protein Surface plasmon resonance was used to the plasminogen binding ability of SEN in a was a and SEN was to over the for such a SEN binding occurred at a that was by mass and to Site-directed mutagenesis was to examine the role of lysine residues in the structural integrity and of GAS residues for analysis based on their known role in plasminogen binding by GAS SEN and SENΔ434–435) A. Song Y.P. Parikh S. Fischetti V.A. Pancholi V. Infect. Immun. 2004; 72: 94-105Crossref PubMed Scopus (96) Google or S. pneumoniae α-enolase (SENK252 + (33Bergmann S. Wild D. Diekmann O. Frank R. Bracht D. Chhatwal G.S. Hammerschmidt S. Mol. 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PubMed Scopus Google Scholar). of α-enolase enzymatic activity and plasminogen binding ability by and surface plasmon resonance was used for functional comparison of SEN mutants with the Enzymatic activity was retained for SENK252 + and was reduced in SENK334A and and was abolished in SENK344E and mutant forms of SEN with human that all mutants plasminogen except for and A and the plasminogen binding ability of the SEN mutants in was surface plasmon and SENK334A to wild SENK344E plasminogen binding This is to the of binding less from mass and the of the binding also this The ability to bind human was for SENK252 + 255A, SENK435L, and SEN These that plasminogen with both lysine + 255 and 434 + in GAS The crystal structure of S. pneumoniae determined at 2 revealed an octameric structure (34Ehinger S. Schubert S. Hammerschmidt S. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). 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In the we and to the structural of SEN and mutant an a of and structural of the for protein on it was except for SENK344E, all SEN as The SENK344E mutant was to a of and the in as by is with a of the of this mutant and could account for the which was greater than SEN and each of the other These structural in the SENK344E mutant also in between the lysine-binding kringle domains of plasminogen and for the plasminogen binding capacity for this SEN The ability of SEN to bind plasminogen has been demonstrated for GAS and other bacterial species (27Pancholi V. Fischetti V.A. J. Biol. Chem. 1998; 273: 14503-14515Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, S. Wild D. Diekmann O. Frank R. Bracht D. Chhatwal G.S. Hammerschmidt S. Mol. Microbiol. 2003; 49: 411-423Crossref PubMed Scopus (191) Google Scholar). 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