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, Fö rster resonance energy transfer (FRET) was used to investigate the 10 lM 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 nearphysiological 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.

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

“…In agreement with our findings, previous EM studies by Nishida et al found a proportion of C3b molecules in this alternative conformation (25). In addition, recent FRET data obtained for C3b in complex with SCR1-4 from FH suggested some degree of mobility of the TED domain (26). Large displacements of the CUB-TED region seem feasible as these take place during the structural transition from C3 to C3b (4) and also from C3b to iC3b (27).…”

Structure of complement C3(H2O) revealed by quantitative cross-linking/mass spectrometry and modeling

“…The Positioning of TED in C3(H 2 O) Versus C3b-Of note, two of the five C3(H 2 O)/C3b-mutual, MG1-TED cross-links correspond to residue-pairs that are far apart in the crystal structure of C3b (PDB 2I07); 38.3 and 40.4 Å, respectively, compared with a theoretical cross-linking limit of 27.4 Å (Experimental Procedures). This could be explained if the TED domains of C3(H 2 O) and C3b were both mobile with respect to MG1 in solution, as also suggested by previous studies (25,54,55). Our data additionally suggest differences between C3(H 2 O) and C3b with respect to the organization of the CUB and TED domains relative to MG1.…”

Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling

“…Of note, two of the five C3(H 2 O)/C3b-mutual, MG1-TED cross-links correspond to residue-pairs that are far apart in the crystal structure of C3b (PDB 2I07 ); 38.3 and 40.4 Å, respectively, compared with a theoretical cross-linking limit of 27.4 Å (Experimental Procedures). This could be explained if the TED domains of C3(H 2 O) and C3b were both mobile with respect to MG1 in solution, as also suggested by previous studies ( 25 , 54 , 55 ). Our data additionally suggest differences between C3(H 2 O) and C3b with respect to the organization of the CUB and TED domains relative to MG1.…”