Abstract
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 alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) 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 alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). 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.
Highlights
Honorary Research Fellow. 3 A Royal Society University Research Fellow. 4 A Royal Society Professor. 5 An Australian Research Council (ARC) Australian Professorial Fellow. 6 An NHMRC R
We demonstrate that the plasminogen-binding motif residues Lys252 and Lys255 and the C-terminal Lys434 and Lys435 residues are located adjacently in the group A Streptococcus (GAS) surface enolase (SEN) structure and play a concerted role in the binding of human plasminogen
Characterization of Wild-type GAS SEN—The structure of wild-type SEN was examined by means of in silico modeling, mass spectrometry (MS), and far-UV Circular Dichroism (CD) spectroscopy
Summary
Honorary Research Fellow. 3 A Royal Society University Research Fellow. 4 A Royal Society Professor. 5 An ARC Australian Professorial Fellow. 6 An NHMRC R. We first constructed an in silico model of octameric GAS SEN (Fig. 1A) based on the known crystal structure of S. pneumoniae ␣-enolase [34].
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