The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin1,2. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn’s disease and ischaemia-reperfusion injuries1. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer’s disease has been identified3. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-540115 and NDT95137276, have been identified by C5a radioligand-binding assays4. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp2135.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.
Subscribe to Journal
Get full journal access for 1 year
only $3.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Woodruff, T. M., Nandakumar, K. S. & Tedesco, F. Inhibiting the C5-C5a receptor axis. Mol. Immunol. 48, 1631–1642 (2011)
Klos, A., Wende, E., Wareham, K. J. & Monk, P. N. International Union of Basic and Clinical Pharmacology. Complement peptide C5a, C4a, and C3a receptors. Pharmacol. Rev. 65, 500–543 (2013)
Landlinger, C. et al. Active immunization against complement factor C5a: a new therapeutic approach for Alzheimer’s disease. J. Neuroinflammation 12, 150 (2015)
Hutchison, A. J. & Krause, J. E. The discovery of small molecule C5a antagonists. Annu. Rep. Med. Chem. 39, 139–147 (2004)
Sumichika, H. et al. Identification of a potent and orally active non-peptide C5a receptor antagonist. J. Biol. Chem. 277, 49403–49407 (2002)
Brodbeck, R. M. et al. Identification and characterization of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a novel, orally bioavailable C5a receptor inverse agonist. J. Pharmacol. Exp. Ther. 327, 898–909 (2008)
Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA 105, 877–882 (2008)
Robertson, N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36–44 (2011)
Oswald, C. et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature 540, 462–465 (2016)
White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012)
Zhang, D. et al. Two disparate ligand-binding sites in the human P2Y1 receptor. Nature 520, 317–321 (2015)
Cheng, R. K. Y. et al. Structural insight into allosteric modulation of protease-activated receptor 2. Nature 545, 112–115 (2017)
Jazayeri, A. et al. Extra-helical binding site of a glucagon receptor antagonist. Nature 533, 274–277 (2016)
Lu, J. et al. Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40. Nat. Struct. Mol. Biol. 24, 570–577 (2017)
Waters, S. M. et al. Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction. J. Biol. Chem. 280, 40617–40623 (2005)
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)
Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011)
Tehan, B. G., Bortolato, A., Blaney, F. E., Weir, M. P. & Mason, J. S. Unifying family A GPCR theories of activation. Pharmacol. Ther. 143, 51–60 (2014)
Floyd, D. H. et al. C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast. J. Biol. Chem. 278, 35354–35361 (2003)
Klco, J. M., Lassere, T. B. & Baranski, T. J. C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor. J. Biol. Chem. 278, 35345–35353 (2003)
Rabiet, M. J., Huet, E. & Boulay, F. Complement component 5a receptor oligomerization and homologous receptor down-regulation. J. Biol. Chem. 283, 31038–31046 (2008)
Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007)
Hebert, T. E. et al. A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. 271, 16384–16392 (1996)
Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)
Manglik, A. et al. Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012)
Wu, H. et al. Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012)
Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013)
Higginbottom, A. et al. Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site. J. Biol. Chem. 280, 17831–17840 (2005)
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protocols 4, 706–731 (2009)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)
Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016)
Martyna, G. J., Klein, M. L. & Tuckerman, M. Nosé-Hoover chains: the canonical emsemble via continuous dynamics. J. Chem. Phys. 97, 2635–2643 (1992)
Martyna, G. J., Tobias, D. J. & Klein, M. L. Constant pressure molecular dynamics algorithms. J. Chem. Phys. 101, 4177–4189 (1994)
Kräutler, V., van Gunsteren, W. F. & Hunenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22, 501–508 (2001)
Tuckerman, M., Berne, B. J. & Martyna, G. J. Reversible multiple time scale molecular dynamics. J. Chem. Phys. 97, 1990–2001 (1992)
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996)
We thank R. Owen, J. Waterman and D. Axford for technical support. We thank C. G. Tate and other colleagues at Heptares Therapeutics for suggestions and comments.
The authors are employees of Heptares Therapeutics and are shareholders of Sosei Group, the parent company of Heptares. Heptares is a drug discovery and development company working in the field of G-protein-coupled receptor structure-based drug design.
Reviewer Information Nature thanks A. Christopoulos, I. Kufareva and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Figure 1 Comparison of wild-type and thermostabilized C5aR1 and the C5aR1 StaR crystallization construct in schematic representation.
a, Thermal stability of C5aR1 measured using [3H]NDT9513727 binding after solubilization in DDM. Wild-type full-length C5aR1 (closed circles) has a melting temperature (Tm) of 18 °C ± 1.05 °C, and C5aR1 StaR full-length (open circles) has a Tm of 44 °C ± 0.7 °C. Data are mean ± s.d. from 3 independent experiments. b, C5aR1 StaR crystallization construct in schematic snake plot representation. Thermostabilizing mutations (green) are: S85A, I91A, I142A, N146R, L156A, F172A, R232A, A234E, L311E, S317E and N321E. Residues forming the NDT9513727 pocket are coloured pink. Disordered residues in the structure are grey. The disulfide bond between Cys1093.25 and Cys188 is denoted by a dashed yellow line. c, Multiple sequence alignment of human, chimpanzee, orangutan, gorilla, macaque, gerbil, cattle, mouse, rat and trout C5aR1 across TM5. The asterisk indicates the tryptophan residue at Ballesteros–Weinstein position 5.49 that is crucial for the interaction of the small-molecule NDT9513727 with C5aR1.
a, Competition assays by displacement of 125I-radiolabelled C5a (125I-C5a), by C5a, NDT9513727 and W54011 applied to membranes from HEK293T cells transiently expressing full-length (1–350) wild-type C5aR1. b, Competition assays by displacement of 125I-C5a, by C5a, NDT9513727 and W54011 applied to membranes from HEK293T cells transiently expressing the full-length (1–350) C5aR1 StaR. Data are mean ± s.e.m. from three biologically independent experiments c, Calculated pIC50 values. Data are representative of three independent experiments ± s.e.m. d–f, 2D chemical structures of the small-molecule C5aR1 antagonists NDT9513727 (d), NDT9520492 (e) and W54011 (f).
a, b, Typical C5aR1 StaR(30–333) non-fusion crystals grown in lipidic cubic phase and complexed with NDT9513727, shown in visible light (a), and under polarized light (b). c, Crystals displayed diffraction out to approximately 2.5 Å after exposure to a non-attenuated beam for 0.07 s per 0.25 degrees of oscillation at beamline I24, Diamond Light Source, UK. d–f, Views of C5aR1 StaR(30–333) packing in the monoclinic crystal system P1211, along the a (d), b (e) and c (f) axis.
Extended Data Figure 4 Cold competition of wild-type C5aR1 and C5aR1 StaR (29–333) bound to [3H]NDT9513727.
a–f, Cold competition of 200 nM [3H]NDT9513727 to solubilized cell lysate containing wild-type C5aR1 or C5aR1 StaR (29–333) with either NDT9513727, C5a agonist, PMX53 or W-54011. Data are representative of four independent experiments performed in duplicate ± s.d. IC50 values inset with s.d. in parentheses. The datasets for the C5a peptide and PMX53 could not be analysed owing to absent competition. g, Data are mean ± s.e.m. from four biologically independent experiments performed in duplicate. The mean pIC50 values (s.e.m. in brackets) are 6.27 (0.10), 6.75 (0.23), 6.58 (0.10) and 6.54 (0.19) for the competition of NDT9513727 and W-54011 against wild-type and then C5aR1 StaR (29–333), respectively. Dagger symbol denotes no value owing to lack of observed competition.
a, Molecular dynamics simulation of wild-type C5aR1 with NDT9513727 over a 250-ns time course monitoring the root mean square deviation (r.m.s.d.) of all NDT9513727 heavy atoms. Inset, molecular dynamics model at time point 250 ns. b, Molecular dynamics simulation of C5aR1 (W213L) with NDT9513727 over a 200-ns time course monitoring the r.m.s.d. of all NDT9513727 heavy atoms. Inset, molecular dynamics model at time point 200 ns.
a, Saturation binding of [3H]NDT9513727 to solubilized cell lysate containing wild-type C5aR1. b, Single experiment showing fluorescence size-exclusion analysis of solubilized cell lysates containing indicated mutant variants of C5aR1 with a C-terminal green fluorescent protein (GFP) tag, in 1% digitonin. c–h, Saturation binding of [3H]NDT9513727 to solubilized cell lysates containing indicated mutant variants of C5aR1. Data are representative from three biologically independent experiments performed in duplicate ± s.d. Kd values are inset with s.d. in parentheses. The mean pKd values (s.e.m. in brackets) are 6.59 (0.08), 6.42 (0.2), 6.28 (0.21) and 6.65 (0.15) for the wild-type, L125F, T129L and T217L mutants, respectively. The datasets for A128F, T129F and W213L could not be analysed unambiguously owing to marked loss of specific binding.
Extended Data Figure 7 Detailed view of the C5aR1 non-crystallographic dimer and ligand-binding interface.
a, Schematic of ligand–protomer interactions in the extra-helical NDT9513727-binding site across the C5aR1 non-crystallographic dimer. Colour scheme of the boxes is as in Fig. 1b. b, Close-up structural view of interactions depicted in a. c–k, Chainbow representation of the C5aR1 asymmetric unit (from a view parallel to the membrane, and rotated 90° to view with cylindrical helices from extracellular space) compared to a representative subset of crystallographic and non-crystallographic GPCR dimeric assemblies present in the PDB. l, The C5aR1 non-crystallographic dimer reported here most closely resembles that previously postulated for the SMO receptor.
Monitoring interhelical distances between TM3-5 and TM4-5 in C5aR with NDT9513727 bound across a 250ns time course in molecular dynamics simulation.
Monitoring interhelical distances between TM3-5 and TM4-5 in C5aR with NDT9513727 bound across a 250ns time course in molecular dynamics simulation. (MP4 9123 kb)
Monitoring interhelical distances between TM3-5 and TM4-5 in C5aR with no ligand bound across a 250ns time course in molecular dynamics simulation
Monitoring interhelical distances between TM3-5 and TM4-5 in C5aR with no ligand bound across a 250ns time course in molecular dynamics simulation. (MP4 7896 kb)
About this article
Cite this article
Robertson, N., Rappas, M., Doré, A. et al. Structure of the complement C5a receptor bound to the extra-helical antagonist NDT9513727. Nature 553, 111–114 (2018) doi:10.1038/nature25025
Bioorganic & Medicinal Chemistry (2019)
Journal of Chemical Information and Modeling (2019)
Journal of Biological Chemistry (2019)
Scientific Reports (2019)