title: Dynamic structural changes during complement C3 activation analyzed by hydrogen/deuterium exchange mass spectrometry.
abstract: Proteolytic cleavage of component C3 to C3b is a central step in the activation of complement. Whereas C3 is largely biologically inactive, C3b is directly involved in various complement activities. While the recently described crystal structures of C3 and C3b provide a molecular basis of complement activation, they do not reflect the dynamic changes that occur in solution. In addition, the available C3b structures diverge in some important aspects. Here we have utilized hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to investigate relative changes in the solution-phase structures of C3 and C3b. By combining two forms of mass spectrometry we could maximize the primary sequence coverage of C3b and demonstrate the feasibility of this method for large plasma proteins. While the majority of the 82 peptides that could be followed over time showed only minor alterations in HDX, we observed clear changes in solvent accessibility for 16 peptides, primarily in the alpha-chain (alpha'NT, MG6-8, CUB, TED, C345C domains). Most of these peptides could be directly linked to the structural transitions visible in the crystal structures and revealed additional information about the probability of the structural variants of C3b. In addition, a discontinuous cluster of seven peptides in the MG3, MG6, LNK and alpha'NT domains showed a decreased accessibility after activation to C3b. Although no gross conformational changes are detected in the crystal structure, this area may reflect a structurally flexible region in solution that contributes to C3 activation and function.
authors.affiliation: Department of Medicine, Division of Rheumatology, University of Pennsylvania, Philadelphia, PA USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Schuster
authors.forename: Daniel
authors.initials: D
authors.lastname: Ricklin
authors.forename: Krisztián
authors.initials: K
authors.lastname: Papp
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Molnar
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Coales
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Georgia
authors.initials: G
authors.lastname: Sfyroera
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Michael S
authors.initials: MS
authors.lastname: Winters
authors.forename: John D
authors.initials: JD
authors.lastname: Lambris
mesh_terms: D000595:Amino Acid Sequence; D003167:Complement Activation; D003176:Complement C3 / Q000737:chemistry*; D003179:Complement C3b / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003176:Complement C3; D010455:Peptides; D003179:Complement C3b