C5aR2 signaling mechanisms via the β-arrestin-dependent pathway observed in microsecond-scale molecular dynamics...

The complement system primarily regulates innate immunity, contributing to nonspecific antipathogen defense, and is linked to adaptive immunity. One of its components, C5 convertase, initiates the last stage of the complement cascade, leading to the cleavage of C5 into C5a and C5b and the formation of the membrane attack complex, which causes cell lysis. In addition, C5a acts as a proinflammatory mediator by regulating leukocyte chemotaxis. C5a binds to 2 G protein-coupled receptors: C5aR1 and C5aR2. C5aR1 activation is involved in inflammation through both G protein and β-arrestin-mediated signaling, while the effects of C5a binding to C5aR2 evoking only the β-arrestin signaling pathway are less defined. C5aR2, often considered a decoy receptor, exhibits both proinflammatory and anti-inflammatory effects in various disease models. To elucidate the physiological significance and therapeutic potential of C5aR2 signaling, we performed 4 μs-scale molecular dynamics simulations of the C5a-C5aR2-β-arrestin 1 complex starting from the active-state β-arrestin conformation. Two possible helix 8 (H8) conformations found in Protein Data Bank structures of C5aR1 used as templates for C5aR2 were tested. The canonical, outward-directed H8, lacking an amphipathic amino acid composition, untangled in all simulations. The noncanonical, inverted conformation of H8 remained stable in these 4-μs-long simulations, with a slight, incomplete clockwise rotation and the simultaneous outward movement of TM7, as observed in sweet-taste G protein-coupled receptors. Nevertheless, in both simulation systems, a short hydrophobic H8 fragment (LPAA) formed a stable anchor to the lipid bilayer throughout the 4-μs simulation. Based on our observations, we suggest that H8 could be limited to this short helical hydrophobic LPAA anchor that stabilizes the active-state β-arrestin conformation. These findings may explain the distinct, non-G protein-mediated signaling of C5aR2 compared with C5aR1 and provide insights into the 2 C5a-mediated signaling pathways, which exhibit significantly different therapeutic potential in the treatment of inflammatory diseases. SIGNIFICANCE STATEMENT: This extensive 4-μs-long, 6-replica molecular dynamics study provides important insights into the molecular basis of C5aR2 signaling, which can lead to either proinflammatory or anti-inflammatory effects, and could be exploited in the treatment of inflammatory diseases. To best of the authors' knowledge, it is the first study to present the molecular determinants of β-arrestin-biased signaling in the C5aR2 system. In addition, it elucidates the molecular mechanisms underlying β-arrestin-mediated signaling, focusing on its conformations and interactions with the receptor.