It is well established that high levels of fetal hemoglobin (HbF) can overcome the pathophysiologic defects in sickle cell disease (SCD) and beta-thalassemia. Despite the recent success of gene therapy in increasing HbF in SCD and the usefulness of hydroxyurea in improving patient outcomes, there remains a pressing need for more effective small molecules to treat the vast majority of SCD patients who will not benefit from gene therapy in the foreseeable future. In addition to the strong negative regulatory transcription factors BCL11A (Sankaran et al, 2008) and ZBTB7A(LRF) (Masuda et al, 2016), the MBD2 NuRD complex, which recognizes and binds at sites of CpG methylation, is essential for HBG silencing in adult erythroid cells (Rupon et al, 2006; Yu et al, 2019; Shang et al, 2023). It has been shown previously that depletion of CHD4, a critical component of MBD2-NuRD which binds to the core complex through GATAD2A (Gnanapragasam et al, 2011), by as little as 50% induces high levels of HbF without blocking differentiation in primary adult human erythroid cells (Amaya et al, 2013; Lan et al, 2021; Vinjumar et al, 2021). To identify potential targets for small-molecule disruption of MBD2-NuRD, we employed the AlphaFold3 ML program to predict the structure of the protein-protein interaction between GATAD2A and CHD4 that tethers the latter to the MBD2-NuRD complex. This analysis indicated that a small helical region in the CR-2 domain of GATAD2A binds to the interface between the C-terminal domains 1 and 2 (CTD1/2) of CHD4. Intracellular NanoBRET assays confirmed strong binding between the entire CR-2 region or the small helix from this region and CHD4(CTD12) domains. Isothermal calorimetry likewise demonstrated tight binding between these domains in vitro. Furthermore, mutating amino acids in the CR-2 helix, which are predicted to be critical for the interaction, disrupted high-affinity binding to CHD4 (CTD1/2). Enforced expression of wild-type GATAD2A, but not GATAD2A mutated at these critical amino acids, resulted in silencing of HBG expression in HUDEP-2 cells in which GATAD2A was knocked out. Prime editing (PE) of the same critical amino acids in the endogenous GATAD2A(CR-2) region resulted in over 40% HBG/(HBG+HBB) RNA levels and likewise over 40 % HbF levels. Immunoprecipitation assays showed dissociation of CHD4 from GATAD2A in the edited cells. NOME SEQ assays demonstrated the same loss of nucleosomes over the HBG promoter as seen in MBD2 knockout cells, and chromatin immunoprecipitation assays showed markedly decreased occupancy of CHD4. RNA seq analysis was carried out to identify other genes affected by the PE editing. HBG1, HBG2, and BGLT3 were upregulated, and HBB was mildly downregulated, while neither CD235 nor CD71, markers of erythroid differentiation, were affected. Lentiviral vector-mediated expression of the small wild-type CR-2 peptide characterized in binding assays resulted in over 15% HBG/(HBG+HBB) RNA levels and dissociation of GATAD2A from CHD4, while in contrast expression of the corresponding peptide with mutations of critical amino acids at the interface with CHD4 (CTD1/2) did not affect either. Together, these experiments demonstrate an on-target effect of disrupting the GATAD2A interaction with CHD4 to relieve MBD2-NuRD mediated silencing of HBG expression. In summary, structural and biophysical studies guided by AlphaFold3 predictions and followed by functional genetic, biochemical, and cellular assays showed that a small peptide can disrupt the interaction between GATAD2A and CHD4 in the MBD2-NuRD complex, thereby inducing high levels of Hb F in adult human erythroid cells. Given the viability and mild phenotype of MBD2 knockout mice and the clinical success of multiple peptidomimetic drugs, these results identify a specific target for the development of small molecules to therapeutically induce high levels of Hb F in patients with SCD and beta-thalassemia.

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.

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 β-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.

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 sug-gested as possible exceptions to 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 no single factor dictating charge selectivity, demonstrat-ing the challenge to predict such interactions. Further, readers that bind both cationic and neutral ligands display an unprece-dented change in mechanism: binding Kme3 via cation−π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery challenges traditional understanding of molecular recognition of tetraalkylammo-niums by aromatic cages in myriad protein-ligand interactions and establishes a new framework for selective inhibitor design by exploiting differences in charge-dependence.

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.

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 ( p BrPrF) 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.

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.

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.

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.

The development of novel bioconjugation strategies is useful for various therapeutic and diagnostic applications. This work describes the synthesis and genetic encoding of an unnatural amino acid that serves as a functional handle for numerous bioconjugations. In particular it can be employed in bioorthogonal [2+2+2] cycloaddition reactions to either conjugate a small-molecule probe/drug or to immobilize a protein on a surface. More information can be found in the communication by D. D. Young et al. on page 310 in Issue 3, 2020 (DOI: 10.1002/cbic.201900391).

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.

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.

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.