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

The interaction of hC5a with C5aR, previously hypothesized to involve a "two-site" binding, (i) recognition of the bulk of hC5a by the N-terminus (NT) of C5aR ("site1"), and (ii) recognition of C-terminus (CT) of hC5a by the extra cellular surface (ECS) of the C5aR ("site2"). However, the pharmacological landscapes of such recognition sites are yet to be illuminated at atomistic resolution. In the context, unique model complexes of C5aR, harboring pharmacophores of diverse functionality at the "site2" has recently been described. The current study provides a rational illustration of the "two-site" binding paradigm in C5aR, by recruiting the native agonist hC5a and engineered antagonist hC5a(A8). The hC5a-C5aR and hC5a(A8)-C5aR complexes studied over 250 ns of molecular dynamics (MD) each in POPC bilayer illuminate the hallmark of activation mechanism in C5aR. The intermolecular interactions in the model complexes are well supported by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) based binding free energy calculation, strongly correlating with the reported mutational studies. Exemplified in two unique and contrasting molecular complexes, the study provides an exceptional understanding of the pharmacological divergence observed in C5aR, which will certainly be useful for search and optimization of new generation "neutraligands" targeting the hC5a-C5aR interaction.

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

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.

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

The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure-function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a ((h)C5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure-function data, it is clear that unlike the mouse C5a ((m)C5a), the (h)C5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in (h)C5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in (h)C5a, by exploring the inherent conformational dynamics in (m)C5a, (h)C5a and in its point mutants, including the proteolytic mutant des-Arg(74)-(h)C5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg(74)-Tyr(23) and Arg(37)-Phe(51) "cation-π" pairs as the molecular "allosteric switches" on (h)C5a that potentially functions as a damper of C5aR signaling.

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

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 ß-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.