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

Methylation of arginine (Arg) residues on histones creates a new binding epitope, enabling recognition by aromatic cage binding pockets in Tudor domains; these protein-protein interactions (PPIs) govern gene expression. Despite their biological importance, the molecular details of methylated Arg recognition are poorly understood. While the desolvation, hydrogen bonding, and guanidinium stacking of methylated Arg have been explored in model systems and proposed to contribute to binding, direct interactions between the methyl groups and the aromatic residues in the binding pocket have not previously been investigated. Herein, we mechanistically study the CH3-π interactions between the SPIN1 triple Tudor domain and histone asymmetric dimethylarginine. We find that these CH3-π interactions are electrostatically tunable, exhibiting cation-π character, albeit attenuated relative to cation-π interactions with quaternary ammonium ions, offering key insight into how methylation of Arg alters its binding epitope to enable new PPIs.

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

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.

WDR5 Binding to Histone Serotonylation Is Driven by an Edge-Face Aromatic Interaction with Unexpected Electrostatic Effects.

Histone serotonylation has emerged as a key post-translational modification. WDR5 preferentially binds to serotonylated histone 3 (H3), and this binding event has been associated with tumorigenesis. Herein, we utilize genetic code expansion, structure-activity relationship studies, and computation to study an edge-face aromatic interaction between WDR5 Phe149 and serotonin on H3 that is key to this protein-protein interaction. We find experimentally that this edge-face aromatic interaction is unaffected by modulating the electrostatics of the face component but is weakened by electron-withdrawing substituents on the edge component. Overall, these results elucidate that this interaction is governed by van der Waals forces as well as electrostatics of the edge ring, a result that clarifies discrepancies among previous theoretical models and model system studies of this interaction type. This is the first evaluation of the driving force of an edge-face aromatic interaction at a protein-protein interface and provides a key benchmark for the nature of these understudied interactions that are abundant in the proteome.

Stimulus-Induced Relief of Intentionally Incorporated Frustration Drives Refolding of a Water-Soluble Biomimetic Foldamer.

Frustrated, or nonoptimal, interactions have been proposed to be essential to a protein's ability to display responsive behavior such as allostery, conformational signaling, and signal transduction. However, the intentional incorporation of frustrated noncovalent interactions has not been explored as a design element in the field of dynamic foldamers. Here, we report the design, synthesis, characterization, and molecular dynamics simulations of the first dynamic water-soluble foldamer that, in response to a stimulus, exploits relief of frustration in its noncovalent network to structurally rearrange from a pleated to an intercalated columnar structure. Thus, relief of frustration provides the energetic driving force for structural rearrangement. This work represents a previously unexplored design element for the development of stimulus-responsive systems that has potential application to materials chemistry, synthetic biology, and molecular machines.

Development of Multivalent Conjugates with a Single Non-Canonical Amino Acid.

Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.

More Than π-π-π Stacking: Contribution of Amide-π and CH-π Interactions to Crotonyllysine Binding by the AF9 YEATS Domain.

Lysine crotonylation (Kcr) is a histone post-translational modification that is implicated in numerous epigenetic pathways and diseases. Recognition of Kcr by YEATS domains has been proposed to occur through intermolecular amide-π and alkene-π interactions, but little is known about the driving force of these key interactions. Herein, we probed the recognition of lysine crotonylation and acetylation by the AF9 YEATS domain through incorporation of noncanonical Phe analogs with distinct electrostatics at two positions. We found that amide-π interactions between AF9 and acyllysines are electrostatically tunable, with electron-rich rings providing more favorable interactions. This differs from trends in amide-heteroarene interactions and provides insightful information for therapeutic design. Additionally, we report for the first time that CH-π interactions at Phe28 directly contribute to AF9's recognition of acyllysines, illuminating differences among YEATS domains, as this residue is not highly conserved but has been shown to impart selectivity for specific post-translational modification.

Genetic Encoding of a Bioconjugation Handle for [2+2+2] Cycloaddition Reactions.

Protein bioconjugates have many critical applications, especially in the development of therapeutics. Consequently, the design of novel methodologies to prepare protein bioconjugates is of great importance. Herein we present the development and optimization of a novel strategy to prepare bioconjugates through a genetically encoded [2+2+2] cycloaddition reaction. To do this, a novel unnatural amino acid (UAA) containing a dipropargyl amine functionality was synthesized and incorporated site specifically. This UAA-containing protein was reacted with an alkyne-containing fluorophore to afford a covalently linked, well-defined protein bioconjugate. This reaction is convenient with an optimized reaction time of just two hours at room temperature and yields a stable, polysubstituted benzene ring. Overall, this work contributes a new bioconjugation strategy to the growing toolbox of reactions to develop protein bioconjugates, which have a myriad of applications.

Mechanistic investigation and further optimization of the aqueous Glaser-Hay bioconjugation.

The Glaser-Hay bioconjugation has recently emerged as an efficient and attractive method to generate stable, useful bioconjugates with numerous applications, specifically in the field of therapeutics. Herein, we investigate the mechanism of the aqueous Glaser-Hay coupling to better understand optimization strategies. In doing so, it was identified that catalase is able to minimize protein oxidation and improve coupling efficiency, suggesting that hydrogen peroxide is produced during the aqueous Glaser-Hay bioconjugation. Further, several new ligands were investigated to minimize protein oxidation and maximize coupling efficiency. Finally, two novel strategies to streamline the Glaser-Hay bioconjugation and eliminate the need for secondary purification have been developed.

Utilization of alkyne bioconjugations to modulate protein function.

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

Probing the non-covalent forces key to the thermodynamics of ß-hairpin unfolding.

Although it is well understood that the graph of the free energy of unfolding (ΔG) of a globular protein with temperature approximates to a negative parabola, there is as yet no link between this global (G) ΔG G(T) function and the individual structural elements-residue type and the non-covalent forces between groups-contributing to it. As such, there is little understanding of how each structural element contributes to the globally assessed changes of enthalpy (ΔH G), entropy (ΔS G), and heat capacity (ΔC p(G)) of unfolding calculated from the ΔG G(T) function. To address this situation, we consider here an alternative approach to examining fold stability. Specifically, we examine the local (L) reporting of the thermodynamics of unfolding provided by each residue. By using 1H NMR spectroscopy to monitor the response of the individual mainchain amide N-H groups of ß-hairpin peptides with temperature, we generate local ΔG L(T) functions, using these to calculate the local enthalpy (ΔH L), entropy (ΔS L), and heat capacity (ΔC p(L)) of unfolding. Mapping the thermodynamic changes in this way, for specific point-mutations, provides new information about how specific residues, non-covalent forces, and secondary structure type, contribute to folding. This type of information provides new details of the factors contributing to the typically measured global ΔG G(T) function.

Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain.

Amide-π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide-π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide-π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere-π interaction. We show that compared to the amide-π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.