Structural complexes of the agonist, inverse agonist and antagonist bound C5a receptor: insights into pharmacology and signaling

Abstract

The C5a receptor (C5aR) is a pharmacologically important G-protein coupled receptor (GPCR) that interacts with (h)C5a, by recruiting both the "orthosteric" sites (site1 at the N-terminus and site2 at the ECS, extra cellular surface) on C5aR in a two site-binding model. However, the complex pharmacological landscape and the distinguishing chemistry operating either at the "orthosteric" site1 or at the functionally important "orthosteric" site2 of C5aR are still not clear, which greatly limits the understanding of C5aR pharmacology. One of the major bottlenecks is the lack of an experimental structure or a refined model structure of C5aR with appropriately defined active sites. The study attempts to understand the pharmacology at the "orthosteric" site2 of C5aR rationally by generating a highly refined full-blown model structure of C5aR through advanced molecular modeling techniques, and further subjecting it to automated docking and molecular dynamics (MD) studies in the POPC bilayer. The first series of structural complexes of C5aR respectively bound to a linear native peptide agonist ((h)C5a-CT), a small molecule inverse agonist (NDT) and a cyclic peptide antagonist (PMX53) are reported, apparently establishing the unique pharmacological landscape of the "orthosteric" site2, which also illustrates an energetically distinct but coherent competitive chemistry ("cation-π" vs. "π-π" interactions) involved in distinguishing the established ligands known for targeting the "orthosteric" site2 of C5aR. Over a total of 1 μs molecular dynamics (MD) simulation in the POPC bilayer, it is evidenced that while the agonist prefers a "cation-π" interaction, the inverse agonist prefers a "cogwheel/L-shaped" interaction in contrast to the "edge-to-face/T-shaped" type π-π interactions demonstrated by the antagonist by engaging the F275(7.28) of the C5aR. In the absence of a NMR or crystallographically guided model structure of C5aR, the computational model complexes not only provide valuable insights for understanding the C5aR pharmacology, but also emerge as a promising platform for the design and discovery of future potential drug candidates targeting the (h)C5a-C5aR signaling axes.

The Model Structures of the Complement Component 5a Receptor (C5aR) Bound to the Native and Engineered hC5a

“…However, the mechanism of such action is still unclear in structural terms. In continuation to our earlier reports 8 , 9 , 13 , the comparison of the h C5a-C5aR, h C5a(A8)-C5aR model structural complexes, including the CT peptide variants of h C5a(A8) presented in the study provide the necessary rationalization important for understanding the observed antagonism and the switching of antagonism to agonism at the “site2” of C5aR. Moreover, the native agonist ( h C5a-C5aR) and the engineered antagonist ( h C5a(A8)-C5aR) bound model complexes, respectively presented in the current study rationalize a large set of point mutation based binding and signaling data 12 , 14 – 20 , by estimating the residue specific energetic contribution toward overall binding in structural terms.…”

“…The topologically unique model of C5aR described earlier 8 , 9 , presented in Fig. 1 illustrates a modestly folded β-hairpin like structure with ~30% residues in ordered β-sheet conformation, as estimated from the in silico folding studies of the predicted extended extracellular loop 2 (ECL2) polypeptide [Ac-Y174-RVVREEYFPPKVL C188 GVDYSHDKR-R198-NH 2 ] 8 .…”

“…In our prior studies 9 , we have illustrated the interaction of h C5a-CT peptide at the “site2” of the modeled C5aR with minimum interference from the NT peptide of C5aR. However, to illustrate a “two-site” binding interaction between h C5a and C5aR, it is highly essential to understand the molecular interaction at the “site1” involving the bulk of h C5a and the NT peptide of the C5aR.…”

“…In our quest to understand the h C5a-C5aR interaction better, we recently generated unique structural models of C5aR 8 and subsequently illustrated the plausible orthosteric “site2” on its ECS 9 , by recruiting a variety of functionally diverse small molecule ligands, including the CT peptide ( 64 NISHKDMQLGR 74 ) of h C5a. In the current study, we subjected the modeled C5aR to pilot experimental scrutiny, involving biophysical techniques and further screened the model against the native agonist h C5a 2 (74 amino acids) and the engineered antagonist (73 amino acids) h C5a(A8) 10 .…”

“…In the current study, we subjected the modeled C5aR to pilot experimental scrutiny, involving biophysical techniques and further screened the model against the native agonist h C5a 2 (74 amino acids) and the engineered antagonist (73 amino acids) h C5a(A8) 10 . Objective was to decipher the plausible orthosteric “site1” on the NMR derived NT peptide 11 , grafted to the modeled C5aR 9 for generating the first set of distinct model molecular complexes, precisely illustrating the pharmaceutical landscape of the “two-site” binding paradigm in C5aR. Though, both h C5a and h C5a(A8) share ~90% sequence identity, h C5a(A8) competitively binds to the C5aR, albeit weakly (IC 50 ~ 35 nM) compared to h C5a (IC 50 ~ 3 nM) for reasons clearly not described 12 .…”