Cascade activation of the complement system is one of the key mechanisms of the immune system against pathogenic infections. It is a complex network of plasma proteins, including inflammatory peptides, proteases, and integral membrane receptors that function together in a synergistic manner. Once the complement system is activated, it effectively removes microorganisms by forming a membrane attack complex (MAC). The complement system is cleaved by different proteases to produce a number of peptides, and these complement factors activate the corresponding receptors or effectors. Abnormally activated complement systems are closely associated with the development of various diseases, such as immunodeficiency and autoimmune disorders. In this process, the peptide fragments C3a and C5a produced from the cleavage of complement C3 and C5 amplify the immune response by recruiting immune cells, also known as anaphylatoxins.
C3a and C5a function mainly by activating their responsive receptors C3aR and C5aR1/C5aR2, which are typical G protein-coupled receptors. Under agonist activation, C3aR can act through different signaling pathways such as downstream Gi/Gq/βarrs, whereas C5aR1 is also able to activate the downstream Gi and βarrs signaling pathways, and C5aR2 is unable to activate the G-protein signaling pathway, but only the βarrs signaling pathway.
In addition, human C3a and C5a contain 77 and 74 amino acids, respectively, and previous studies have shown that they can activate the corresponding receptors through a two-site binding mode and associate with the N-terminal end of the receptor, the second extracellular loop (ECL), and the periplasmic core. In addition, the C-terminal hydrolysis of C3a and C5a produces peptides that can also activate the corresponding receptors, such as the peptides EP54 and EP67 produced by the hydrolysis of C5a, as well as the artificially designed peptide C5apep. Meanwhile, the last amino acids of C3a and C5a can be physiologically regulated to be excised to produce C3ades-Arg and C5ades-Arg, which have a reduced ability to activate the corresponding receptors. Although researchers understand the function of C3a and C5a in activating the corresponding receptors, little is known about their ligand-recognition mechanisms.
On October 17, 2023, Arun K. Shukla and his collaborator Ramanuj Banerjee from IIT, along with Cornelius Gati’s team from the University of Southern California, published their latest research results in Cell under the title “Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors”. They analyzed the complexes of C3aR or C5aR1 with downstream G proteins in the presence of ligand activation by cryo-electron microscopy. They revealed the binding modes of the corresponding ligand-activated receptors. In addition, the researchers have also analyzed the structural basis for the activation of the receptors by C3ades-Arg, C5ades-Arg, and a biased agonist, providing a detailed molecular basis for the development of drugs targeting C3aR and C5aR1/2 in the corresponding diseases.
The researchers first examined the activation of downstream signaling pathways by C3aR as well as C5aR1 in the presence of different ligands. The researchers found that EP54 from C3a was able to activate the Gi signaling pathway and βarr1/2 signaling pathway of C3aR. Human-derived C5apep and C5a were able to activate mC5aR1 and inhibit cAMP, although they were slightly less potent in activating mC5aR1 than in activating hC5aR1. In addition, C5a is a full agonist of mC5aR1, whereas C5apep is only partially agonistic for hC5aR1 and mC5aR1.
C3a and C5a share 35% sequence homology, and they both consist of four helices linked together by disulfide bonds, as well as a terminal arginine residue located at the tail end of the helix (Arg77 in C3a and Arg74 in C5a). Through structural analysis and base mutation activity detection, the researchers confirmed that both C3a-C3aR and C5a-C5aR1 have a two-site model, in which one site of the ligand binds to the ECL2 of the receptor, and the second site interacts with multiple amino acid residues in the membrane-penetrating structural domain. In addition, the N-terminal domain of C5aR1 is also involved in C5a binding. While the ECL2 of C3aR is long, functional experiments show that a large part of it is not related to C3a binding.
Upon binding to the receptor, C3a and C5a retained their helical folded structures, but also underwent some changes from the original structures, such as 30° and 45° tilting of the H3 helices of C3a and C5a. In addition, the distal carboxyl terminus of C3a underwent a significant rotation, whereas the distal carboxyl terminus of C5a changed from a short helix in the basal state to an extended conformation. The overall position of C3a in C3aR differs dramatically from that of C5a in C5aR1, with the globular structural domains of C3a and C5a both tilted 120° relative to their distal carboxyl terminus. Unlike the C3a and C5a, which are oriented in opposite directions on the outside of the receptor at an angle of approximately 90°, the carboxyl termini of C3a and C5a, on the other hand, form a hook-like conformation that buries them in a binding pocket.
Both C3a and C5a have a natural modulation of their activity by removing the last arginine via a hydrolyzing enzyme, resulting in a diminished ability to activate the corresponding receptor.
In summary, the researchers resolved the structures of the complexes of C3aR and C5aR1 with downstream G proteins under multiple ligand activation scenarios by cryo-electron microscopy and analyzed the activation mechanisms, as well as the naturally occurring mechanisms of activity regulation and the molecular mechanisms of biased agonist activation of C3aR. This provides a theoretical basis for understanding the molecular mechanisms by which the complement functions.
References：
1. Yadav, Manish K., et al. “Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.” Cell 186.22 (2023): 4956-4973.
2. Hawksworth, Owen A., et al. “New concepts on the therapeutic control of complement anaphylatoxin receptors.” Molecular immunology 89 (2017): 36-43.
3. Pandey, Shubhi, et al. “Emerging insights into the structure and function of complement C5a receptors.” Trends in biochemical sciences 45.8 (2020): 693-705.