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, 286 (36), 31661-75

Structural Model of ligand-G Protein-Coupled Receptor (GPCR) Complex Based on Experimental Double Mutant Cycle Data: MT7 Snake Toxin Bound to Dimeric hM1 Muscarinic Receptor

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Structural Model of ligand-G Protein-Coupled Receptor (GPCR) Complex Based on Experimental Double Mutant Cycle Data: MT7 Snake Toxin Bound to Dimeric hM1 Muscarinic Receptor

Catherine Marquer et al. J Biol Chem.

Abstract

The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554-1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409-420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 Å apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.

Figures

FIGURE 1.
FIGURE 1.
Identification of extracellular loops of hM1 receptor involved in MT7 binding. A, schematic representations of the various chimeric receptors constructed by exchanging the extracellular loops between the hM1 and hM3 receptors colored in black and gray, respectively. B, sequence alignments of the extracellular loops E1, E2, and E3 of the hM1 and hM3 receptors. C, IC50(mut)/IC50(WT) values of MT7 toxin on wild-type hM1 and hM3 and chimeric muscarinic receptors. D, ribbon representation of an hM1 structural model indicating the extracellular loops E1, E2, and E3 colored in cyan, magenta, and orange, respectively. The same colors were used to differentiate the three extracellular loops in A, B, and C. N-ter, N terminus; C-ter, C terminus.
FIGURE 2.
FIGURE 2.
Double mutant cycle analysis of MT7-hM1 complex. Values of ΔΔGint (reported in Table 2) determined from changes in affinity constants are presented in colored bars. The different blue colors correspond to loop I; the different yellow, orange, and red colors correspond to loop II; and the different green colors correspond to loop III of MT7. Inset, ribbon representation of MT7 indicating the loops involved in the binding. Blue, red, and green, MT7 loops I, II, and III, respectively.
FIGURE 3.
FIGURE 3.
Identification of dimer interface by two first independent docking calculations. The random search and systematic grid search are on the left and right, respectively. A and B, variation of restraint energy as a function of the global r.m.s. deviation. The global r.m.s.d. was calculated using as reference the structure with the lowest restraint energy and by fitting the Cα atoms of MT7 and hM1 dimer excluding the extracellular loops. Superimpositions of the backbone of the 10 lowest restraint energy structures are shown: C and D, extracellular view; E and F, transverse view; G and H, intracellular view. The TM6 and TM7 domains are colored in orange and yellow, respectively. I and J, basa (in Å2) at the hM1 dimer interface calculated on complexes obtained by random and grid searches, respectively. (Energy thresholds of 40 and 35 kcal/mol were used for random and grid searches, respectively.)
FIGURE 4.
FIGURE 4.
Structural model of MT7-dimeric hM1 complex based on experimental double mutant cycle data. A, restraint energy as a function of r.m.s.d. from the MT7-hM1 dimer for about 30,000 calculated complexes obtained by five independent docking simulations (supplemental Table 1). The reference structure was the lowest energy structure. The r.m.s.d. was calculated using the Cα atoms of MT7 and hM1 dimer excluding the extracellular loops. B, histogram of the averaged minimum distances between residue pairs with ΔΔGint >0.7 kcal/mol and involved in ambiguous distance restraints (a) and with ΔΔGint <0.7 kcal/mol and not used in the docking calculations (b). C and D, two perpendicular views of a superimposed ribbon representation of the 10 best structures. E and F, two perpendicular views of a schematic of the lowest restraint energy model. hM1A, hM1B, and MT7 are in red, blue, and green, respectively. hM1 loops are in cyan except E2, which is in magenta.
FIGURE 5.
FIGURE 5.
Average buried accessible surface area (in Å2) in 10 structures of MT7-dimeric hM1 complex. A, MT7 residues at the interface with the hM1 receptor; arrows indicate the β-sheets of MT7. B, hM1 residues at the interface with MT7. C, hM1 residues located at the dimer interface. White rectangles symbolize the hM1 TM helices. In each plot, hM1A is at the top, and hM1B at the bottom. The average buried accessible surface area is calculated on the best MT7-dimeric hM1 structures (see text). The error bars indicate the r.m.s. associated with the average values.
FIGURE 6.
FIGURE 6.
Specific side chain-side chain interactions in 10 structures of MT7-dimeric hM1 complex. A, tip of the MT7 loop II-hM1A. B, tip of the MT7 loop I-hM1B. C, MT7 loops II and III-hM1A E2. D, MT7 loop I-hM1B E1. hM1A, hM1B, hM1 loop E1, hM1 loop E2, and toxin MT7 are colored in red, blue, cyan, magenta, and green, respectively.
FIGURE 7.
FIGURE 7.
TM6/TM7 dimerization interface in 10 structures of MT7-hM1 dimer complex. hM1A and hM1B are displayed in red and blue schematics, respectively. The transverse view (A) and extracellular view (B) are on the left and right, respectively. Three contacts at the interface are supported by the same residues on both protomers: residues Leu-376, Leu-399, and Leu-406 are colored in orange, green, and yellow, respectively. The cluster of Ile-383 of hM1A and Leu-402 and Trp-405 of hM1B and the cluster of Ile-383 of hM1B and Leu-402 and Trp-405 of hM1A are colored in pale cyan and cyan, respectively. The cluster of Leu-372 of hM1A and Ile-413, Cys-417, and Leu-420 of hM1B is colored in hot pink, and the cluster of Leu-372 of hM1B and Ile-413, Cys-417, and Leu-420 of hM1A is colored in violet.
FIGURE 8.
FIGURE 8.
TM interface in MT7-hM1 dimer complex is determined by distance between MT7 loops. A, ribbon representation of MT7 in red with “hot spot” residues Arg-34, Trp-10, and Arg-52 in green. The MT7 principal axis and the orthogonal projection of this axis on the membrane plane are displayed in pink. B, hM1 dimer conformation calculated from our experimental data viewed from the extracellular face (Tyr-179/Trp-400 in green). C, hM1 dimer conformation based on the CXCR4 chemokine structure viewed from the extracellular face (Tyr-179/Trp-400 in yellow). D, measurements of the distances between the two centers of gravity of the protomers of the receptor (○) and the two centers of mass of the MT7 binding sites (Tyr-179/Trp-400) of the protomers (■) during the step by step rotation of each protomer around their vertical axis. Distances are averaged on 10 structures, and the error bars indicate the r.m.s. associated with the average values. Under the graph, the TM involved in the dimer interface is indicated, and in two extreme cases, the localization of the MT7 binding sites in the corresponding dimer configuration is displayed. The hM1 receptor is symbolized by a gray oval, and the MT7 binding site is symbolized by a white circle.

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