Trimethyllysine Reader Proteins Exhibit Widespread Charge-Agnostic Binding via Different Mechanisms to Cationic and Neutral Ligands

Abstract

In the last 40 years, cation−π interactions have become part of the lexicon of noncovalent forces that drive protein binding. Indeed, tetraalkylammoniums are universally bound by aromatic cages in proteins, suggesting that cation−π interactions are a privileged mechanism for binding these ligands. A prominent example is the recognition of histone trimethyllysine (Kme3) by the conserved aromatic cage of reader proteins, dictating gene expression. However, two proteins have recently been suggested as possible exceptions to the conventional understanding of tetraalkylammonium recognition. To broadly interrogate the role of cation−π interactions in protein binding interactions, we report the first large-scale comparative evaluation of reader proteins for a neutral Kme3 isostere, experimental and computational mechanistic studies, and structural analysis. We find unexpected widespread binding of readers to a neutral isostere with the first examples of readers that bind the neutral isostere more tightly than Kme3. We find that no single factor dictates the charge selectivity, demonstrating the challenge of predicting such interactions. Further, readers that bind both cationic and neutral ligands differ in mechanism: binding Kme3 via cation−π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery explains apparently contradictory results in previous studies, challenges traditional understanding of molecular recognition of tetraalkylammoniums by aromatic cages in myriad protein−ligand interactions, and establishes a new framework for selective inhibitor design by exploiting differences in charge dependence.

Contribution of Electrostatic CH3–π Interactions to Recognition of Histone Asymmetric Dimethylarginine by the SPIN1 Triple Tudor Domain

“…While a clear trend is observed, binding of wild-type protein (Tyr at residue 98) shows tighter binding than expected based on its ESP value. As we have observed previously, Hammett σ para values result in a better fit for Tyr on the LFER plot . Additionally, the tighter than expected binding could potentially be due to the hydroxyl group of Tyr98 forming favorable water-mediated hydrogen bonds with the side chain and backbone of H3R8me2a (Figure S7).…”

“…Nonetheless, the variation in binding, amounting to about 1 kcal/mol in binding affinity, or 5-fold difference for SPIN1, simply based on the electrostatics of the aromatic ring, is significant. We observe a slope of −0.04 ± 0.01 for Rme2a CH 3 –π interactions, which is smaller in magnitude than slopes for Kme3 cation−π interactions of comparable distances and geometries (ranging from −0.06 to −0.13), − quantitatively demonstrating that the Rme2a CH 3 –π interaction is less sensitive to changes in electrostatics than the Kme3 CH 3 –π interaction. This may be due to greater delocalization of the charge in methylated arginine versus methylated lysine.…”

“…To determine the contribution of CH 3 –π interactions in the SPIN1 TTD’s binding of Rme2a, we incorporated a series of noncanonical para -substituted phenylalanine amino acids using genetic code expansion at the Y98 position This approach enables variation of the electrostatics of a single aromatic residue within a reader protein to quantitatively assess the driving force of binding to a PTM . We have previously utilized this technique to evaluate several histone PTMs, including Kme3, binding inside aromatic pockets, − ,, and others have used similar approaches to evaluate ligand binding for other biologically relevant molecules, such as acetylcholine. − However, this strategy has not previously been employed to evaluate binding of Rme by aromatic boxes. We identified Y98 as an optimal residue for investigation, as the face of the aromatic ring is 3.5 Å from a methyl group of Rme2a (Figure , PDB 4MZF).…”

“…In addition to reducing the desolvation cost, the methyl groups themselves often appear to directly interact with the aromatic residues in binding pockets (Figure b). , However, the contribution and driving force for these CH 3 –π interactions of Rme have not been explored previously. In addition to the hydrophobic effect and dispersion forces, the CH 3 –π interaction may also be driven by electrostatic interactions between the CH 3 (δ+) arising from the cationic nature of arginine and the quadrupole of the aromatic ring, resulting in cation−π character similar to that of the cation−π interaction between Kme3 and aromatic residues. ,− However, the charge delocalization of the Rme guanidinium is a key difference between this moiety and tetraalkylammoniums like Kme3, thus meriting further investigation into the nature of guanidinium CH 3 –π interactions.…”