Solution Insights Into the Structure of the Efb/C3 Complement Inhibitory Complex as Revealed by Lysine Acetylation and Mass Spectrometry

J Am Soc Mass Spectrom. 2008 Jan;19(1):55-65. doi: 10.1016/j.jasms.2007.10.009.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Chromatography, High Pressure Liquid
  • Complement C3 / chemistry*
  • Lysine / chemistry*
  • Molecular Sequence Data
  • Nanotechnology
  • Peptide Mapping
  • Recombinant Proteins
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Tandem Mass Spectrometry*

Substances

  • Bacterial Proteins
  • Complement C3
  • Efb protein, Staphylococcus aureus
  • Recombinant Proteins
  • Lysine