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 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.
From the School of Biological Sciences, Wollongong NSW 2522, Australia.
Amanda J
AJ
Cork
School of Chemistry, University of Wollongong, Wollongong NSW 2522, Australia.
Slobodan
S
Jergic
Department of Genetics of Microorganisms, Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University of Greifswald, Greifswald D-17487, Germany.
Sven
S
Hammerschmidt
School of Molecular and Microbial Sciences and Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia.
Bostjan
B
Kobe
Department of Pathology, Ohio State University, Columbus, Ohio 43210.
Vijay
V
Pancholi
Department of Chemistry, University of Cambridge, Cambridge CB2 1TN, United Kingdom.
Justin L P
JLP
Benesch
Department of Chemistry, University of Cambridge, Cambridge CB2 1TN, United Kingdom.
Carol V
CV
Robinson
School of Chemistry, University of Wollongong, Wollongong NSW 2522, Australia.
Nicholas E
NE
Dixon
From the School of Biological Sciences, Wollongong NSW 2522, Australia.
J Andrew
JA
Aquilina
From the School of Biological Sciences, Wollongong NSW 2522, Australia. Electronic address: mwalker@uow.edu.au.
Mark J
MJ
Walker
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; C112984:streptococcal surface enolase; D010751:Phosphopyruvate Hydratase; D008239:Lysine