Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring

The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 β-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts. ### Competing Interest Statement GRA, HP and RKJ are inventors on patents describing the hC3Nb2 nanobody and the EWEuH fusion protein. HGO is co-founder of Commit Biologics that has licensed these patents