Dennis A. Dougherty

Cation-π Interactions in Chemistry and Biology: A New View of Benzene, Phe, Tyr, and Trp

Cations bind to the π face of an aromatic structure through a surprisingly strong, noncovalent force termed the cation-π interaction. The magnitude and generality of the effect have been established by gas-phase measurements and by studies of model receptors in aqueous media. To first order, the interaction can be considered an electrostatic attraction between a positive charge and the quadrupole moment of the aromatic. A great deal of direct and circumstantial evidence indicates that cation-π interactions are important in a variety of proteins that bind cationic ligands or substrates. In this context, the amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) can be viewed as polar, yet hydrophobic, residues.

Acetylcholine binding by a synthetic receptor: implications for biological recognition

The neurotransmitter acetylcholine (ACh) is bound with 50-micromolar affinity by a completely synthetic receptor (host) comprising primarily aromatic rings. The host provided an overall hydrophobic binding site, but one that could recognize the positive charge of the quaternary ammonium group of ACh through a stabilizing interaction with the electron-rich pi systems of the aromatic rings (cation-pi interaction). Similar interactions may be involved in biological recognition of ACh and other choline derivatives.

Cis – trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-Hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2–M3), can link binding to gating through a cis–trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis–trans energy gap of the proline analogue and the activation of the channel, suggesting that cis–trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2–M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2–M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

Cation-π interactions in ligand recognition and catalysis

The cation-pi interaction is a potent, general noncovalent binding force that is observed in a wide range of biological contexts. Here, we present an overview of well documented cases in which a cation-pi interaction makes an important contribution to small-molecule recognition at a protein binding site. From these and other studies it is clear that, in addition to the hydrophobic effect, hydrogen bonding and ion pairing, the cation-pi interaction must be considered when evaluating drug-receptor interactions.

A Gating Charge Transfer Center in Voltage Sensors

Open and Closed Case Voltage-dependent ion channels are gated by voltage sensors that show a switchlike response to voltage differences across the membrane. Tao et al. (p. 67; see the cover) used mutagenesis, electrophysiology, and x-ray crystallography to gain insight into the molecular basis of this response in voltage-dependent potassium channels. An occluded site was identified that catalyzes translation of positive charges across the membrane. The closed channel appears to be associated with a distribution of conformations, depending on the degree of hyperpolarization of the membrane, whereas the open channel appears to be associated with a specific conformation. Thus, the transition of the ion channel from open to closed occurs over a very small voltage difference. An occluded site stabilizes charged amino acids as they cross the membrane field to achieve switchlike channel opening. Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic “cap” and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

Unnatural amino acids as probes of protein structure and function

Nonsense suppression methodology, for incorporating unnatural amino acids into proteins, has enabled a wide range of studies into protein structure and function using both in vitro and in vivo translation systems. Although methodological challenges remain, scores of unnatural amino acids have been employed that include both subtle and dramatic variants of the natural set. A number of insights that would not have been possible using conventional site-directed mutagenesis have been gained.

Nicotine binding to brain receptors requires a strong cation–π interaction

Nicotine addiction begins with high-affinity binding of nicotine to acetylcholine (ACh) receptors in the brain. The end result is over 4,000,000 smoking-related deaths annually worldwide and the largest source of preventable mortality in developed countries. Stress reduction, pleasure, improved cognition and other central nervous system effects are strongly associated with smoking. However, if nicotine activated ACh receptors found in muscle as potently as it does brain ACh receptors, smoking would cause intolerable and perhaps fatal muscle contractions. Despite extensive pharmacological, functional and structural studies of ACh receptors, the basis for the differential action of nicotine on brain compared with muscle ACh receptors has not been determined. Here we show that at the α4β2 brain receptors thought to underlie nicotine addiction, the high affinity for nicotine is the result of a strong cation–π interaction to a specific aromatic amino acid of the receptor, TrpB. In contrast, the low affinity for nicotine at the muscle-type ACh receptor is largely due to the fact that this key interaction is absent, even though the immediate binding site residues, including the key amino acid TrpB, are identical in the brain and muscle receptors. At the same time a hydrogen bond from nicotine to the backbone carbonyl of TrpB is enhanced in the neuronal receptor relative to the muscle type. A point mutation near TrpB that differentiates α4β2 and muscle-type receptors seems to influence the shape of the binding site, allowing nicotine to interact more strongly with TrpB in the neuronal receptor. ACh receptors are established therapeutic targets for Alzheimer’s disease, schizophrenia, Parkinson’s disease, smoking cessation, pain, attention-deficit hyperactivity disorder, epilepsy, autism and depression. Along with solving a chemical mystery in nicotine addiction, our results provide guidance for efforts to develop drugs that target specific types of nicotinic receptors.

In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system

A general method for the incorporation of unnatural amino acids into ion channels and membrane receptors using a Xenopus oocyte expression system has been described. A large number of unnatural amino acids have been incorporated into the nAChR, GIRK, and Shaker K+ channels. Continuing efforts focus on incorporating unnatural amino acids that differ substantially from the natural amino acids, for example, residues that include fluorophores. In addition, we are addressing the feasibility of incorporating unnatural amino acids into ion channels and membrane receptors in mammalian cells.

Backbone Mutations in Transmembrane Domains of a Ligand-Gated Ion Channel: Implications for the Mechanism of Gating

An approach to identify backbone conformational changes underlying nicotinic acetylcholine receptor (nAChR) gating was developed. Specific backbone peptide bonds were replaced with an ester, which disrupts backbone hydrogen bonds at the site of mutation. At a conserved proline residue (alphaPro221) in the first transmembrane (M1) domain, the amide-to-ester mutation provides receptors with near-normal sensitivity, although the natural amino acids tested other than Pro produce receptors that gate with a much larger EC50 than normal. Therefore, a backbone hydrogen bond at this site may interfere with normal gating. In the alphaM2 domain, the amide-to-ester mutation yielded functional receptors at 15 positions, 3 of which provided receptors with >10-fold lower EC50 than wild type. These results support a model for gating that includes significant changes of backbone conformation within the M2 domain.

Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery.

The acronym SePhaChARNS, for “selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry,” is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes “upregulation” of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning. The chaperone is a pharmacological one, nicotine; and the chaperoned molecules are α4β2* nAChRs. SePhaChARNS may also underlie two inadvertent therapeutic effects of tobacco use: (1) the inverse correlation between tobacco use and Parkinson’s disease; and (2) the suppression of seizures by nicotine in autosomal dominant nocturnal frontal lobe epilepsy. SePhaChARNS arises from the thermodynamics of pharmacological chaperoning: ligand binding, especially at subunit interfaces, stabilizes AChRs during assembly and maturation, and this stabilization is most pronounced for the highest-affinity subunit compositions, stoichiometries, and functional states of receptors. Several chemical and pharmacokinetic characteristics render exogenous nicotine a more potent pharmacological chaperone than endogenous acetylcholine. SePhaChARNS is modified by desensitized states of nAChRs, by acid trapping of nicotine in organelles, and by other aspects of proteostasis. SePhaChARNS is selective at the cellular, and possibly subcellular, levels because of variations in the detailed nAChR subunit composition, as well as in expression of auxiliary proteins such as lynx. One important implication of the SePhaChARNS hypothesis is that therapeutically relevant nicotinic receptor drugs could be discovered by studying events in intracellular compartments rather than exclusively at the surface membrane.