Model structures of inactive and peptide agonist bound C5aR: Insights into agonist binding, selectivity and activation

Abstract

C5a receptor (C5aR) is one of the major chemoattractant receptors of the druggable proteome that binds C5a, the proinflammatory polypeptide of complement cascade, triggering inflammation and SEPSIS. Here, we report the model structures of C5aR in both inactive and peptide agonist (YSFKPMPLaR; a=D-Ala) bound meta-active state. Assembled in CYANA and evolved over molecular dynamics (MD) in POPC bilayer, the inactive C5aR demonstrates a topologically unique compact heptahelical bundle topology harboring a β-hairpin in extracellular loop 2 (ECL2), derived from the atomistic folding simulations. The peptide agonist bound meta-active C5aR deciphers the “site2” at an atomistic resolution in the extracellular surface (ECS), in contrast to the previously hypothesized inter-helical crevice. With estimated Ki≈2.75 μM, the meta-active C5aR excellently rationalizes the IC50 (0.1–13 μM) and EC50 (0.01–6 μM) values, displayed by the peptide agonist in several signaling studies. Moreover, with Ki≈5.3×105 μM, the “site2” also illustrates selectivity, by discriminating the stereochemical mutant peptide (YSFkPMPLaR; k=D-Lys), known to be inert toward C5aR, up to 1 mM concentration. Topologically juxtaposed between the structures of rhodopsin and CXCR1, the C5aR models also display excellent structural correlations with the other G-protein coupled receptors (GPCRs). The models elaborated in the current study unravel many important structural insights previously not known for regulating the agonist binding and activation mechanism of C5aR.

Allosterism in human complement component 5a (hC5a): a damper of C5a receptor (C5aR) signaling

“…It is noteworthy that the NT of CXCR4 also interacts with the core of vMIP-II that demonstrates an appreciable conformational change in its loop and NT region (Liwang, Wang, Sun, Peiper, & Liwang, 1999;Qin et al, 2015), post binding to CXCR4. On the other hand, the linear peptide agonist [YSFKPMPLaR; a = D-Ala; based on the CT peptide of h C5a]-bound meta-active model structure of C5aR (Rana & Sahoo, 2015) also indicates a single contiguous binding site on the extra cellular surface of C5aR (lacks 26 residues on NT) similar to CXCR4, further justifying a similar binding interaction between the CT of h C5a and C5aR (Rana & Sahoo, 2015). Thus, it is quite likely that h C5a-C5aR interaction will also demonstrate a similar two-site 1:1 stoichiometry, as observed for CXCR4 receptor (Qin et al, 2015), by involving both the CT and the core of h C5a.…”

“…As evidenced, the Arg 74 -Tyr 23 "cation-π" interaction appears weak in Arg 37 /Ala, moderate in Arg 37 /Lys, and strong in Arg 37 /Asp mutant, compared to wild-type h C5a (Figure 8). This suggests that increase in the helical conformation of helix 3 (Supplementary Figure S12) allosterically strengthens the Arg 74 -Tyr 23 "cation-π" interaction ( Figure 8), which alternatively restricts the availability of the Arg 74 for properly docking the h C5a to C5aR (Rana & Sahoo, 2015), potentially contributing toward the loss of C5aR signaling. On the other hand, the Arg 74 on h C5a appears flexible (Supplementary Figure S3(c)) over the duration of MD and this inherent flexibility could be very important for the functional regulation of h C5a.…”

“…Data were plotted in GraphPad Prism (version 6 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com). MD simulation of the h C5a and its variants were carried out at 300 K for over 50 ns each, following the procedures described previously (Joshi, Rana, Wangikar, & Durani, 2006;Rana & Sahoo, 2015). Briefly, the polar amino acids such as Lys and Arg were positively charged, whereas amino acids such as Asp and Glu were negatively charged, in all the systems, which were appropriately neutralized, by randomly placing requisite number of ions to the box prior to the energy minimization.…”

“…Production MD was initiated thereafter, and the trajectory was sampled at 5 ps intervals. "Cation-π" interactions were calculated implementing the following cutoff (d ≤ 6 Å; θ < 90°) as described elsewhere (Marshall, Steele, Thanthiriwatte, & Sherrill, 2009;Rana & Sahoo, 2015). Briefly, the "cation-π" angles were obtained by recruiting the in-house program, which takes PDB files as input and then calculates the angle between the two normal, respectively, projected on the centroids of the aromatic ring and the guanidinium group of the arginine.…”

“…The h C5a-C5aR interaction is of therapeutic importance, as it elicits a plethora of physiological responses ranging from chemoattraction, multiple organ failure, ischemia, and reperfusion injury to apoptosis (Guo & Ward, 2005). The complete molecular interaction is not structurally established yet, though a highly refined model structure of C5aR in complex with a linear peptide agonist based on the C-terminus (CT) of h C5a has recently been described in the literature (Rana & Sahoo, 2015). It is well established that the mutation in the CT region of h C5a Toth et al, 1994), including the des-Arg 74 -h C5a -mimic of the proteolytic mutant of h C5a, produced in plasma - (Cain, Coughlan, & Monk, 2001;Higginbottom et al, 2005) down regulates both C5aR binding and signaling.…”