Human C3a and C3a desArg anaphylatoxins have conserved structures, in contrast to C5a and C5a desArg

Abstract

Complement is a part of innate immunity that has a critical role in the protection against microbial infections, bridges the innate with the adaptive immunity and initiates inflammation. Activation of the complement, by specific recognition of molecular patterns presented by an activator, for example, a pathogen cell, in the classical and lectin pathways or spontaneously in the alternative pathway, leads to the opsonization of the activator and the production of pro-inflammatory molecules such as the C3a anaphylatoxin. The biological function of this anaphylatoxin is regulated by carboxypeptidase B, a plasma protease that cleaves off the C-terminal arginine yielding C3a desArg, an inactive form. While functional assays demonstrate strikingly different physiological effects between C3a and C3a desArg, no structural information is available on the possible conformational differences between the two proteins. Here, we report a novel and simple expression and purification protocol for recombinant human C3a and C3a desArg anaphylatoxins, as well as their crystal structures at 2.3 and 2.6 Å, respectively. Structural analysis revealed no significant conformational differences between the two anaphylatoxins in contrast to what has been reported for C5a and C5a desArg. We compare the structures of different anaphylatoxins and discuss the relevance of their observed conformations to complement activation and binding of the anaphylatoxins to their cognate receptors.

Figure 1

Biological activity of recombinant C3a and C3a desArg. (A) Elution profile of C3a (blue) and C3a desArg (light blue) from the Source 15S column. (B) Comparison of plasma purified and recombinant human C3a and C3a desArg in an N-acetyl-β-d-glucosaminidase-release assay performed on stably transfected RBL cells expressing C3aR.

Figure 2

General overview of the anaphylatoxins structures. (A) Overall structure of human C3a at 2.3 Å resolution. The N- and C-terminal residues are indicated. The four alpha helices are numbered. The three disulfide bridges stabilizing the four-helix bundle are shown in yellow sticks. (B) Structure of the two C3a molecules present in the asymmetric unit and related by noncrystallographic twofold symmetry. (C and D) 2mF_o-DF_c electron density maps, contoured at 1σ for C3a (panel C) and C3a desArg (panel D), respectively. The C3a and C3a desArg molecules are oriented as in panel A.

Figure 3

Comparison of the free and C3-bound anaphylatoxins structures (A) Superimposition of the C3a (violet) and C3a desArg (blue) structures determined in this study. The regions displaying the most significant structural differences are labeled with residue numbers. (B) Superimposition of the recombinant C3a (violet, this study) and the C3a moiety from the human C3 (orange, PDB ID 2A7320). The regions displaying the most significant structural differences are indicated by black lines and labeled with residue numbers.

Figure 4

Comparison of C3a and C3a desArg with other anaphylatoxins. (A) Superimposition of the four-helix bundle fold of the recombinant human C3a (violet, this study), of the C4a moiety from the human C4 (yellow, PDB ID 4FXG32) and of the C5a moiety from the human C5 (green, PDB ID 3CU722). (B) Superimposition of the four-helix-bundle from the C5a moiety of the human C5 (green, PDB ID 3CU722) and from the two monomers of the human C5a desArg (blue, PDB ID 3HQA and cyan, PDB ID 3HQB25), present in the asymmetric unit of the crystal structure, showing the α1-helix swing-out motion of C5a desArg.

Figure 5

Comparison of the interactions stabilizing α1-helix packing within the four-helix bundle for the recombinant human C3a (A) and the human C5a as part of C5 (PDB ID 3CU722) (B). The residues involved in hydrogen bond and salt bridge (dotted lines) formation are highlighted in cyan in the C3a structure, and hydrophobic residues at the α1-α4 helix interface are shown in red in the C5a structure.