Solution insights into the structure of the Efb/C3 complement inhibitory complex as revealed by lysine acetylation and mass spectrometry.
The extracellular fibrinogen-binding protein (Efb), an immunosuppressive and anti-inflammatory protein secreted by Staphylococcus aureus, has been identified as a potent inhibitor of complement-mediated innate immunity. Efb functions by binding to and disrupting the function of complement component 3 (C3). In a recent study, we presented a high-resolution co-crystal structure of the complement inhibitory domain of Efb (Efb-C) bound to its cognate domain (C3d) from human C3 and employed a series of structure/function analyses that provided evidence for an entirely new, conformational change-based mechanism of complement inhibition. To better understand the Efb/C3 complex and its downstream effects on C3 inhibition, we investigated the solvent-accessibility and protein interface of Efb(-C)/C3d using a method of lysine acetylation, proteolytic digestion, and mass spectrometric analysis. Lysine modification in Efb was monitored by the mass increment of lysine-containing fragments. Besides confirming the binding sites observed in co-crystal structure study, the in-solution data presented here suggest additional contacting point(s) between the proteins that were not revealed by crystallography. The results of this study demonstrate that solution-based analysis of protein-protein interactions can provide important complementary information on the nature of protein-protein interactions.
Chen
Hui
H
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Schuster
Michael C
MC
Sfyroera
Georgia
G
Geisbrecht
Brian V
BV
Lambris
John D
JD
D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003176:Complement C3 / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010449:Peptide Mapping; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D003176:Complement C3; C114014:Efb protein, Staphylococcus aureus; D011994:Recombinant Proteins; D008239:Lysine