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Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97.

ABSTRACT: The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA(A) receptor alpha1Phe65, beta2Tyr97, beta2Tyr157, and beta2Tyr205 are present at the beta2/alpha1 interface and have been implicated in forming an important part of the GABA binding site. Here, we have probed interactions of these residues using subtle chemical changes: unnatural amino acid mutagenesis was used to introduce a range of Phe analogs, and mutant receptors expressed in oocytes were studied using voltage-clamp electrophysiology. Serial mutations at beta(2)97 revealed a approximately 20-fold increase in EC50 with the addition of each fluorine atom to a phenylalanine, indicating a cation-pi interaction between GABA and this residue. This is the first example of a cation-pi interaction in loop A of a Cys-loop receptor. Along with previous studies that identified cation-pi interactions in loop B and loop C, the result emphasizes that the location of this interaction is not conserved in the Cys-loop family. The data further show that alpha(1)65 (in loop D) is tolerant to subtle changes. Conversely, mutating either beta2Tyr157 (in loop B) or beta2Tyr205 (in loop C) to Phe substantially disrupts receptor function. Substitution of 4-F-Phe, however, at either position, or 4-MeO-Phe at beta2Tyr157, resulted in receptors with wild-type EC50 values, suggesting a possible hydrogen bond. The molecular scale insights provided by these data allow the construction of a model for GABA docking to the agonist binding site of the GABA(A) receptor.

SUBMITTER: Padgett CL

PROVIDER: S-EPMC2649369 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The chemistry community now recognizes the cation-π interaction as a major force for molecular recognition, joining the hydrophobic effect, the hydrogen bond, and the ion pair in determining macromolecular structure and drug-receptor interactions. This Account provides the author's perspective on the intellectual origins and fundamental nature of the cation-π interaction. Early studies on cyclophanes established that water-soluble, cationic molecules would forego aqueous solvation to enter a hydrophobic cavity if that cavity was lined with π systems. Important gas phase studies established the fundamental nature of the cation-π interaction. The strength of the cation-π interaction (Li(+) binds to benzene with 38 kcal/mol of binding energy; NH4(+) with 19 kcal/mol) distinguishes it from the weaker polar-π interactions observed in the benzene dimer or water-benzene complexes. In addition to the substantial intrinsic strength of the cation-π interaction in gas phase studies, the cation-π interaction remains energetically significant in aqueous media and under biological conditions. Many studies have shown that cation-π interactions can enhance binding energies by 2-5 kcal/mol, making them competitive with hydrogen bonds and ion pairs in drug-receptor and protein-protein interactions. As with other noncovalent interactions involving aromatic systems, the cation-π interaction includes a substantial electrostatic component. The six (four) C(δ-)-H(δ+) bond dipoles of a molecule like benzene (ethylene) combine to produce a region of negative electrostatic potential on the face of the π system. Simple electrostatics facilitate a natural attraction of cations to the surface. The trend for (gas phase) binding energies is Li(+) > Na(+) > K(+) > Rb(+): as the ion gets larger the charge is dispersed over a larger sphere and binding interactions weaken, a classical electrostatic effect. On other hand, polarizability does not define these interactions. Cyclohexane is more polarizable than benzene but a decidedly poorer cation binder. Many studies have documented cation-π interactions in protein structures, where lysine or arginine side chains interact with phenylalanine, tyrosine, or tryptophan. In addition, countless studies have established the importance of the cation-π interaction in a range of biological processes. Our work has focused on molecular neurobiology, and we have shown that neurotransmitters generally use a cation-π interaction to bind to their receptors. We have also shown that many drug-receptor interactions involve cation-π interactions. A cation-π interaction plays a critical role in the binding of nicotine to ACh receptors in the brain, an especially significant case. Other researchers have established important cation-π interactions in the recognition of the "histone code," in terpene biosynthesis, in chemical catalysis, and in many other systems.

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Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97.

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