title: Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism.
abstract: During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104-Glu1032salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,wof monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RGvalues of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.
authors.affiliation: Department of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
authors.forename: Ka Wai
authors.initials: KW
authors.lastname: Fung
authors.affiliation: Department of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
authors.forename: David W
authors.initials: DW
authors.lastname: Wright
authors.affiliation: Department of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
authors.forename: Jayesh
authors.initials: J
authors.lastname: Gor
authors.affiliation: Swann Scientific Consulting Ltd, 110 Sandy Lane, Lymm WA13 9HR, U.K.
authors.forename: Marcus J
authors.initials: MJ
authors.lastname: Swann
authors.affiliation: Department of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, U.K.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Perkins
mesh_terms: D003179:Complement C3b / Q000737:chemistry / Q000378:metabolism; D015935:Complement C4b / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D011487:Protein Conformation / Q000187:drug effects; D000072417:Protein Domains; D012965:Sodium Chloride / Q000494:pharmacology*
chemical_list: D012965:Sodium Chloride; D003179:Complement C3b; D015935:Complement C4b
keywords: complement C4b; dual polarisation interferometry; molecular modelling; small angle X-ray scattering; ultracentrifugation