Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein

Abstract

The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and desensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. We identified unique cooperative binding behavior of a number of 4,6-disubstituted 2-aminopyrimidines to Lymnaea AChBP, with different molecular variants exhibiting positive, nH > 1.0, and negative cooperativity, nH < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine. An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, noncooperative, nicotinic ligands. Positively and negatively cooperative ligands exhibited unique features in the detailed binding determinants and poses of the complexes.

Keywords: allostery; crystallography; nicotinic receptor.

Fig. 1.

Representative titration profiles for 4,6-substituted 2-aminopyrimidine competition with ³H-epibatidine binding showing a range of dissociation constants (K_d) and Hill coefficients (n_H) for ligand binding to Ls-AChBP. Measurements were carried out by a scintillation proximity assay and are reported as an average of at least three individual experiments (±SD).

Fig. 2.

X-ray crystal structures of ligands 32 and 33 (negative cooperativity, n_H < 1) and 15 (positive cooperativity, n_H > 1), in complexes with Ls-AChBP. (A) Radial view of Ls-AChBP pentameric structure in complex with 32. Full occupation of the 10 binding sites in the unit crystal of a dimer of pentamers was evident. A principal, C loop-containing, and complementary face are shown in gray and purple. (B) Expanded radial view of 32 in binding site, including ligand electron density. Side chain carbons of the principal and complementary subunits are shown in gray and purple, respectively. Ligand carbons are in yellow and fluorines in turquoise. (C) Overlay of 32 (blue) and 33 (yellow) crystal structures. Side-chain carbons for 32 are in turquoise. The overlay shows little or no variance in ligand pose or side-chain positions. (D) Superimposition of 15 (yellow) and 33 (blue) crystal structures. Side-chain carbons for 33 are in turquoise. The positively 15 and negatively 32/33 cooperative ligands show a similar positioning of the 4-substituted phenyl rings, but distinct poses or positions for the 2-aminopyrimidine ring and the substituents at position 4 of the pyrimidine ring. Marked changes in the orientation of the side chains of Y185, W53, and Y164 are evident for the positively and negatively cooperative ligands.

Fig. 3.

Superimposition of Ls-AChBP X-ray crystal structures in complex with 33 (A) and 15 (B) with nicotine (PDB code 1UW6) (in orange). 33 and 15 carbons are shown in yellow, and nicotine in orange. The protein side chains are shown in gray for 33 and 15 and pink for nicotine. Both ligands show distinctly different positions from the pyrrolidine and pyridine rings of nicotine, as well as the side-chain positions of residues in the C loop in the principal subunit (Y89 and Y185) and the complementary (W53 and Y164) subunit face.

Fig. 4.

Global differences in X-ray crystal structures of Ls-AChBP bound cooperative ligands in comparison with crystal structure of Ls-AChBP in its Apo form (PDB code 1UX2) and GLIC (PDB code 4NPP). (A) Top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound 15 (in red). Dashed lines (blue and red, respectively) indicate the most significant differences in quaternary structures quantified by measuring distances between T13 backbone α carbon of distant subunits. (B) Superimposition (PyMOL) of Ls-AChBP Apo, chain D (in blue) and 15 complex, chain D (in red). Major differences in the quaternary structures of the AChBP are marked with dashed rectangles (RMS value of ∼0.5 or greater). (C) Chart of differences of Cα distances (n = 5) of diametrical subunits observed in cooperative ligands relative to Apo in comparison with GLIC (GLIC, closed form, is used as a reference). Nicotine used as a control. Observed differences reflect blooming profile of the protein complexes. (D) Plot of differences of Cα dihedral angles (n = 5) observed in cooperative ligands relative to Apo in comparison with GLIC (GLIC closed form used as a reference). Nicotine used as a control. Observed differences reflect twisting profile of the protein complex.