Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism.

Ka Wai Fung,Stephen J Perkins,Jayesh Gor,David W Wright,Marcus J Swann

doi:10.1042/bcj20160744

Ka Wai Fung, Stephen J Perkins + Show 3 more

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https://doi.org/10.1042/bcj20160744

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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-Glu1032 salt 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,w of 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 RG values 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.

Highlights

In the complement system of innate immunity, its activation through the classical and lectin pathways through C4b is distinct from its activation in the alternative pathway through C3b
C4b was shown to be more compact in 50 mM NaCl, when the thioester domain (TED) and MG1 domain are in contact with each other through salt-bridge formation
Using a combination of a sensor surface technology (DPI) and two solution technologies (AUC and small angle X-ray scattering (SAXS)), we showed that this conformational change occurred both at surfaces and in solution

Summary

Introduction

In the complement system of innate immunity, its activation through the classical and lectin pathways through C4b is distinct from its activation in the alternative pathway through C3b. The classical pathway requires binding of C1q to antibody–antigen complexes on pathogen surfaces, whereupon the serine protease C1r2C1s2 is activated [1]. The lectin pathway involves the interaction of mannosebinding lectin to mannose residues on pathogen surfaces, whereupon mannose-binding lectin-associated serine protease (MASP)-1 and MASP-2 are activated [1]. These activated proteases cleave complement C4 (205 kDa) into C4a (9 kDa) and C4b (188 kDa). A salt-bridge interaction between Arg104–Glu1032 is formed, and the reactive thioester bond within the TED is exposed for nucleophilic attack on its targets. Activated C4b is capable of attaching covalently to hydroxyl or amine groups on pathogenic surfaces, acting as an opsonin

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