title: Use of time-resolved FRET to validate crystal structure of complement regulatory complex between C3b and factor H (N terminus).
abstract: Structural knowledge of interactions amongst the ~ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM K(D) (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.
authors.affiliation: EaStCHEM School of Chemistry and Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Isabell C
authors.initials: IC
authors.lastname: Pechtl
authors.forename: Robert K
authors.initials: RK
authors.lastname: Neely
authors.forename: David T F
authors.initials: DT
authors.lastname: Dryden
authors.forename: Anita C
authors.initials: AC
authors.lastname: Jones
authors.forename: Paul N
authors.initials: PN
authors.lastname: Barlow
mesh_terms: D003179:Complement C3b / Q000737:chemistry* / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary
chemical_list: D003179:Complement C3b; D017242:Complement Factor H