Mechanistic studies of a general reaction that decages a wide range of substrates on exposure to visible light are described. The reaction involves a photochemically initiated reduction of a quinone mediated by an appended thioether. After reduction, a trimethyl lock system incorporated into the quinone leads to thermal decaging. The reaction could be viewed as an electron-transfer initiated reduction of the quinone or as a hydrogen abstraction—Norrish Type II—reaction. Product analysis, kinetic isotope effects, stereochemical labeling, radical clock, and transient absorption studies support the electron transfer mechanism. The differing reactivities of the singlet and triplet states are determined, and the ways in which this process deviates from typical quinone photochemistry are discussed. The mechanism suggests strategies for extending the reaction to longer wavelengths that would be of interest for applications in chemical biology and in a therapeutic setting.

Designing subtype-selective agonists for neuronal nicotinic acetylcholine receptors is a challenging and significant goal aided by intricate knowledge of each subtype’s binding patterns. We previously reported that in α6β2 receptors, acetylcholine makes a functional cation−π interaction with Trp149, but nicotine and TC299423 do not, suggesting a distinctive binding site. This work explores hydrogen binding at the backbone carbonyl associated with α6β2 Trp149. Substituting residue i + 1, Thr150, with its α-hydroxy analogue (Tah) attenuates the carbonyl’s hydrogen bond accepting ability. At α6(T150Tah)β2, nicotine shows a 24-fold loss of function, TC299423 shows a modest loss, and acetylcholine shows no effect. Nicotine was further analyzed via a double-mutant cycle analysis utilizing N′-methylnicotinium, which indicated a hydrogen bond in α6β2 with a ΔΔG of 2.6 kcal/mol. Thus, even though nicotine does not make the conserved cation−π interaction with Trp149, it still makes a functional hydrogen bond to its associated backbone carbonyl.

•PNU-120596 does not alter acetylcholine binding interactions•Functional residues in allosteric communication identified•Global network stabilization rather than adjusted agonist binding domainNicotinic acetylcholine receptors (nAChRs) are vital to neuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The α7 nAChR has been implicated in Alzheimer's disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the α7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties, and link PNU-120596, the acetylcholine binding region, and the receptor gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed.Figure optionsDownload full-size imageDownload high-quality image (204 K)Download as PowerPoint slide

Cation–π interactions are common in biological systems, and many structural studies have revealed the aromatic box as a common motif. With the aim of understanding the nature of the aromatic box, several computational methods were evaluated for their ability to reproduce experimental cation–π binding energies. We find the DFT method M06 with the 6-31G(d,p) basis set performs best of several methods tested. The binding of benzene to a number of different cations (sodium, potassium, ammonium, tetramethylammonium, and guanidinium) was studied. In addition, the binding of the organic cations NH4+ and NMe4+ to ab initio generated aromatic boxes as well as examples of aromatic boxes from protein crystal structures were investigated. These data, along with a study of the distance dependence of the cation–π interaction, indicate that multiple aromatic residues can meaningfully contribute to cation binding, even with displacements of more than an angstrom from the optimal cation–π interaction. Progressive fluorination of benzene and indole was studied as well, and binding energies obtained were used to reaffirm the validity of the “fluorination strategy” to study cation–π interactions in vivo.

•A backbone mutation in M2 reveals the importance of an interhelix hydrogen bond•Two noncanonical histidine analogs synthesize and incorporate into GLIC•His234 is a sterically restricted proton-binding site essential for gatingThe cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism.Figure optionsDownload full-size imageDownload high-quality image (185 K)Download as PowerPoint slide

Structures of integral membrane receptors provide valuable models for drug–receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals.

Nicotinic acetylcholine receptors are a diverse set of ion channels that are essential to everyday brain function. Contemporary research studies selective activation of individual subtypes of receptors, with the hope of increasing our understanding of behavioral responses and neurodegenerative diseases. Here, we aim to expand current binding models to help explain the specificity seen among three activators of α4β2 receptors: sazetidine-A, cytisine, and NS9283. Through mutational analysis, we can interchange the activation profiles of the stoichiometry-selective compounds sazetidine-A and cytisine. In addition, mutations render NS9283—currently identified as a positive allosteric modulator—into an agonist. These results lead to two conclusions: (1) occupation at each primary face of an α subunit is needed to activate the channel and (2) the complementary face of the adjacent subunit dictates the binding ability of the agonist.

The publication of the first high-resolution crystal structure of a eukaryotic Cys-loop receptor, GluClα, has provided valuable structural information on this important class of ligand-gated ion channels (LGIC). However, limited functional data exist for the GluCl receptors. Before applying the structural insights from GluCl to mammalian Cys-loop receptors such as nicotinic acetylcholine and GABA receptors, it is important to ensure that established functional features of mammalian Cys-loop receptors are present in the more distantly related GluCl receptors. Here, we seek to identify ligand-binding interactions that are generally associated with Cys-loop receptors, including the frequently observed cation−π interaction. Our studies were performed on the highly homologous GluClβ receptor, because GluClα is not activated by glutamate in Xenopus laevis oocytes. Mutagenesis of the signal peptide and pore lining helix was performed to enhance functional expression and sensitivity to applied ligand, respectively. Conventional and unnatural amino acid mutagenesis indicate a strong cation−π interaction between Y206 and the protonated amine of glutamate, as well as other important ionic and hydrogen bond interactions between the ligand and the binding site, consistent with the crystal structure.

Memantine (Namenda) is prescribed as a treatment for moderate to severe Alzheimer’s Disease. Memantine functions by blocking the NMDA receptor, but the key binding interactions between drug and receptor are not fully elucidated. To determine key binding interactions of memantine, we made side-by-side comparisons of IC50 for memantine and amantadine, a structurally related drug, in the GluN1/GluN2B NMDA receptor. We identified hydrophobic binding pockets for the two methyl groups on memantine formed by the residues A645 and A644 on the third transmembrane helices of GluN1 and GluN2B, respectively. Moreover, we found that while adding two methyl groups to amantadine to produce memantine greatly improves affinity, adding a third methyl group to produce the symmetrical trimethylamantadine diminished affinity. Our results provide a better understanding of chemical-scale interactions between memantine and the NMDA channel, which will potentially benefit the development of new drugs for neurodegenerative diseases involving NMDA receptors.Keywords: amantadine; Memantine; open-channel blocker; unnatural amino acid mutagenesis

Drug-receptor binding interactions of four agonists, ACh, nicotine, and the smoking cessation compounds varenicline (Chantix) and cytisine (Tabex), have been evaluated at both the 2:3 and 3:2 stoichiometries of the α4β2 nicotinic acetylcholine receptor (nAChR). Previous studies have established that unnatural amino acid mutagenesis can probe three key binding interactions at the nAChR: a cation−π interaction, and two hydrogen-bonding interactions to the protein backbone of the receptor. We find that all drugs make a cation−π interaction to TrpB of the receptor. All drugs except ACh, which lacks an N+H group, make a hydrogen bond to a backbone carbonyl, and ACh and nicotine behave similarly in acting as a hydrogen-bond acceptor. However, varenicline is not a hydrogen-bond acceptor to the backbone NH that interacts strongly with the other three compounds considered. In addition, we see interesting variations in hydrogen bonding interactions with cytisine that provide a rationalization for the stoichiometry selectivity seen with this compound.

The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46, and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51, show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic–aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur−π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.

The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

The nicotinic acetylcholine receptors (nAChRs) are a family of closely related but pharmacologically distinct neurotransmitter-gated ion channels. They are therapeutic targets for a wide range of neurological disorders, and a key issue in drug development is selective targeting among the more than 20 subtypes of nAChRs that are known. The present work evaluates a proposed hydrogen bonding interaction involving a residue known as the “loop B glycine” that distinguishes receptors that are highly responsive to ACh and nicotine from those that are much less so. We have performed structure–function studies on the loop B site, including unnatural amino acid mutagenesis, in three different nAChR subtypes and found that the correlation between agonist potency and this residue is strong. Low potency receptor subtypes have a glycine at this key site, and mutation to a residue with a side chain converts a low potency receptor to a high potency receptor. Innately high potency receptors have a lysine at the loop B site and show a decrease in potency for the reverse mutation (i.e., introducing a glycine). This residue lies outside of the agonist binding site, and studies of other residues at the agonist binding site show that the details of how changes at the loop B glycine site impact agonist potency vary for differing receptor subtypes. This suggests a model in which the loop B residue influences the global shape of the agonist binding site rather than modulating any specific interaction.

The serotonin type 3A (5-HT3A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation−π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation−π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation−π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating.Keywords: binding site; Cys-loop; gating; mCPBG; Serotonin; unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis–trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P2105.50, a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures.

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.

The reductive deoxygenation of acyl phosphonates using a Wolff−Kishner-like sequence is described. This transformation allows direct access to alkyl phosphonates from acyl phosphonates at room temperature. The method can be combined with acyl phosphonate synthesis into a one pot, four-step procedure for the conversion of carboxylic acids into alkyl phosphonates. The methodology works well for a variety of aliphatic acids and shows a functional group tolerance similar to that of other hydrazone-forming reactions.

Pharmacophore models for nicotinic agonists have been proposed for four decades. Central to these models is the presence of a cationic nitrogen and a hydrogen bond acceptor. It is now well-established that the cationic center makes an important cation-π interaction to a conserved tryptophan, but the donor to the proposed hydrogen bond acceptor has been more challenging to identify. A structure of nicotine bound to the acetylcholine binding protein predicted that the binding partner of the pharmacophore’s second component was a water molecule, which also hydrogen bonds to the backbone of the complementary subunit of the receptors. Here we use unnatural amino acid mutagenesis coupled with agonist analogs to examine whether such a hydrogen bond is functionally significant in the α4β2 neuronal nAChR, the receptor most associated with nicotine addiction. We find evidence for the hydrogen bond with the agonists nicotine, acetylcholine, carbamylcholine, and epibatidine. These data represent a completed nicotinic pharmacophore and offer insight into the design of new therapeutic agents that selectively target these receptors.

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug–receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K+ channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation–π interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models. Interestingly, no comparable cation–π interaction was found at the aligning residue in the M2 receptor.

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 depression1. 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.

Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure–function relationships, incorporating biophysical probes, and elucidating protein–protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more “orthogonal” to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.

•α6L9′Sβ2L9′SLFM/AAQA nAChR yields high expression levels in Xenopus oocytes.•Expression levels are high enough to permit nonsense suppression studies.•Structure function studies show ACh makes a cation-π interaction with TrpB.•Nicotine, though more potent than ACh, makes no cation-π interaction with TrpB.The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-β2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6β2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure–function studies between Trp149 – a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes – and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6β2 nAChR.Download full-size image

•α4β2α5 receptors modulate rectification properties compared to α4β2 receptors.•α5V9′S allows generation and observation of a homogeneous receptor population.•Mutational analysis shows no functional ligand-binding site at the α5–α4 interface.Nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the α5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an α5 subunit on nAChRs, a method to prepare a homogeneous population of α5-containing receptors must be developed. Here we use a gain of function (9′) mutation to isolate populations of α5-containing nAChRs for characterization by electrophysiology. We find that the α5 subunit modulates nAChR rectification when co-assembled with α4 and β2 subunits. We also probe the α5–α4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the α5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the α5–α4 interface, in contrast to what has recently been demonstrated for the α4–α4 interface in related receptors. In addition, our mutational results suggest that the α5 subunit does not replace the α4 or β2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.

•Unlike 5-HT, mCPBG binds at heteromeric interfaces of the 5-HT3AB receptor.•mCPBG is capable of allosterically modulating serotonin response.•Heteromeric binding sites subtly differ from the homomeric site.•The homomeric binding site contributes disproportionately to receptor activation.The 5-HT3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT3 selective agonist, is capable of binding to and activating the 5-HT3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they conform to a monophasic dose – response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT3AB receptor activation.

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50–60 Å from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (βL9′S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC50 measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors.

Cation–π interactions are common in biological systems, and many structural studies have revealed the aromatic box as a common motif. With the aim of understanding the nature of the aromatic box, several computational methods were evaluated for their ability to reproduce experimental cation–π binding energies. We find the DFT method M06 with the 6-31G(d,p) basis set performs best of several methods tested. The binding of benzene to a number of different cations (sodium, potassium, ammonium, tetramethylammonium, and guanidinium) was studied. In addition, the binding of the organic cations NH4+ and NMe4+ to ab initio generated aromatic boxes as well as examples of aromatic boxes from protein crystal structures were investigated. These data, along with a study of the distance dependence of the cation–π interaction, indicate that multiple aromatic residues can meaningfully contribute to cation binding, even with displacements of more than an angstrom from the optimal cation–π interaction. Progressive fluorination of benzene and indole was studied as well, and binding energies obtained were used to reaffirm the validity of the “fluorination strategy” to study cation–π interactions in vivo.