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 (SEN
K252 + 255A, SEN
K304A, SEN
K334A, SEN
K344E, SEN
K435L, and SEN
Δ434–435) 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 SEN
K344E, 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 SEN
K344E had lost α-enolase activity, which was also reduced in SEN
K334A and SEN
Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN
K252 + 255A, SEN
K435L, and SEN
Δ434–435. 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)
8 is a common bacterial pathogen, causing over 700 million human disease episodes each year (
1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (
1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (
2–
4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (
5–
17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (
3,
17–
19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (
20,
21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μ
m (
22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg
561–Val
562 site (
23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (
20,
23). 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 (
20,
24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α
2-antiplasmin and α
2-macroglobulin (
11,
20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (
6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (
25), the PAM-related protein (
19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (
9,
26), and streptococcal surface enolase (SEN or α-enolase) (
27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (
6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (
27–
29). 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 (
27,
28,
30,
31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (
27,
32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of
Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (
33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of
S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (
34).In this study, we constructed an
in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys
344 plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys
252 and Lys
255 and the C-terminal Lys
434 and Lys
435 residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.
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