Molecular Recognition of Lys and Arg Methylation - American

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Molecular Recognition of Lys and Arg Methylation Joshua E. Beaver, and Marcey L. Waters ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00996 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 26, 2015

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ACS Chemical Biology

Molecular Recognition of Lys and Arg Methylation Joshua E. Beaver*,† and Marcey L. Waters* ‡ Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, North Carolina 27599, United States KEYWORDS: Posttranslational modification, molecular recognition, histone code, Lys and Arg methylation, synthetic receptor, cation-π interaction, epigenetics, reader protein. ABSTRACT: A network of reader proteins and enzymes precisely controls gene transcription through the dynamic addition, removal, and recognition of posttranslational modifications (PTMs) of histone tails. Histone PTMs work in concert with this network to regulate gene transcription through the histone code, and the dysregulation of PTM maintenance is linked to a large number of diseases, including many types of cancer. A wealth of research aims to elucidate the functions of this code, but our understanding of the effects of PTMs, specifically the methylation of lysine (Lys) and arginine (Arg), is lacking. The development of new tools to study PTMs relies on a sophisticated understanding of the mechanisms that drive protein and small molecule recognition in water. In this review, we outline the physical organic concepts that drive the molecular recognition of Lys and Arg methylation by reader proteins and draw comparisons to the binding mechanisms of small molecule receptors for methylated Lys and Arg that have been developed recently.

Human DNA contains over 3 billion base pairs, encodes 20 different amino acids and guides the synthesis of thousands of proteins. Confined to the cell nucleus, DNA is extensively organized into compounding levels of complexity. Double stranded DNA helices are first packaged as nucleosomes by wrapping 146 base pairs around an octet of histone proteins (two each of H2A, H2B, H3, and H4).1 These nucleosomes are further condensed into chromatin, which can be stored in its compact, inactive form, heterochromatin, and wound into chromosomes; or as the less-dense, active form, euchromatin, where it is read, processed, and modified for gene transcription.2,3 A network of enzymes and proteins strictly regulates nucleosome packaging, and in particular, the modification of histone proteins, which is integral in the control of gene transcription. Histone proteins possess unstructured N-terminal tails that protrude from the nucleosome and are heavily modified with posttranslational modifications (PTMs) including acetylation, phosphorylation, ubiquitination, and lysine (Lys) and arginine (Arg) methylation. Histone PTMs can function individually or as a network, known as the histone code,4 to recruit a variety of proteins with diverse functions via binding of “reader” proteins that recognize the PTMs. Binding of reader proteins to specific PTMs (or combinations thereof) results in downstream events, including the addition and removal of additional PTMs, chromatin condensation, or recruitment of transcriptional machinery leading to gene transcription.5,6 Precise control over the dynamic addition and elimination of histone PTMs is critical in governing chromatin structure transcription.7 In particular, the communication, or crosstalk,8 between methylated Lys

and Arg is instrumental in the regulation of gene transcription.9,10 Dysregulation of histone modifications like Lys and Arg methylation has been linked to breast, prostate, colon, bladder, and lung cancers,11 as well as leukemia, cardiovascular diseases, HIV, multiple sclerosis, and spinal muscular atrophy.4,11–15 A deep understanding of the significance of Lys and Arg methylation and their recognition by reader proteins is of paramount importance to the development of inhibitors, probes, and therapeutics for these diseases; however, a comprehensive map of histone PTMs and their function has not been established, despite a surge of research in exploring and translating the histone code. Several techniques are commonly used to probe the influence of histone PTMs on enzyme activity and proteinprotein interactions; however, these PTMs are often low in abundance and are difficult to characterize. Many of the analytical methods for the analysis of protein Lys and Arg methylation currently rely on antibodies. While antibodies offer tight, and often very specific substrate binding, a range of problems can dilute their benefits in molecular recognition of these PTMs.16 Recent studies have demonstrated that adjacent PTMs can negatively affect antibody recognition of the modification of interest, and that some antibodies are unable to distinguish between varying methylation states of Lys on a peptide.17,18 While time-consuming and expensive, mass spectrometry with enrichment is particularly effective for elucidating the presence of neighboring PTMs.19 Other techniques, like on-bead peptide libraries20 and microarrays21– 24 are useful for analyzing protein-PTM interactions and

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the influence of neighboring histone PTMs, but also require intensive screening or the use of antibodies. Synthetic alternatives to antibodies also show promise in the development of new analytical techniques.25 Small molecule receptors have received considerable attention in the development assays to study histone modifications. Many of these studies aim to improve our understanding of the fundamental mechanisms of molecular recognition in water and continue to drive research in the development of novel biomedical applications and materials.26–29 Molecular recognition in water is especially critical in the development of protein inhibitors, drug discovery, sensor assays and the investigation of protein interactions.5,30,31 This review highlights the non-covalent interactions that drive molecular recognition of methylated Lys and Arg on histones and draws analogies to small molecule receptors and their recognition of histone PTMs. Protein Recognition of Methylated Lys The molecular recognition of methylated Lys has received considerable attention for its role in the regulation of gene transcription.9,32–35 From a molecular perspective, it may be surprising at first glance that methylation of Lys provides an epitope for molecular recognition, since the charge on the quaternary ammonium functional group is not changed. However, methylation of Lys has a significant effect on its interaction with water. Lys has a pKa of ~ 10.5 and is protonated under physiological conditions and can be methylated up to three times (Fig. 1).36 Methylation does not affect the overall charge of the residue; however, each subsequent methylation removes a proton from the ε-amine, decreasing its hydrogen bond capacity and increasing hydrophobicity. Perhaps even more surprising, the first x-ray structure of a reader protein (the HP1 chromodomain) bound to KMe3 revealed that the quaternary ammonium sits in what traditionally would be described as a hydrophobic pocket made up of three aromatic residues and only a single carboxylate sidechain. This can be explained by the contribution of cation-π interactions. The cation-π interaction can be described as the coloumbic attraction between a cation and the partially negatively charged πsurface of an aromatic ring, which is arises from the quadrupole moment of the ring.30,37 For quaternary ammonium, the cation-π interaction is primarily driven by this coloumbic attraction, with dispersion and hydrophobic contacts providing additional driving force.30,37–41 Methylation encourages the formation of cation-π interactions between the methylammonium and aromatic surfaces by reducing the desolvation energy of the cation. The ammonium group of unmethylated Lys is too well solvated by water to interact with an aromatic group, but KMe3 is both charged and “greasy”; it cannot hydrogen bond with water, and the charge is distributed over the methyl groups, rather than on nitrogen, resulting in a preferential interaction of the CH3(δ+) with the face of an aromatic ring.42,43 The hydrophobic effect is not the main driver for binding, however. In the case of the HP1 chromodomain, the Waters group was the first to demonstrate the im-

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portance of cation-π interactions to KMe3 recognition. By replacing the nitrogen in KMe3 with an isosteric but neutral carbon (CMe3), they found that the binding affinity is reduced 30-fold, amounting to 2.2 kcal/mol. Using the same approach, the Mecinović group has recently demonstrated that the contribution of cation-π interactions is general among a range of KMe3 reader proteins.44

A

B

C. Figure 1. A. Methylation states of Lys, portraying the decrease in hydrogen-bonding capacity and increase in hydrophobicity as methylation increases. B. Electrostatic potential maps of Lys sidechain methylation states corresponding to those in A. Electrostatic potential maps were generated with MacSpartan: HF/6-21g; isodensity value = 0.02; range = 100 (red, electron rich) to 200 kcal/mol (blue, electron poor). C. Electrostatic potential maps of aromatic sidechains for Phe, Tyr, and Trp, from left to right. Electrostatic potential maps were generated with MacSpartan: HF/6-21g; isodensity value = 0.02; range = -100 (red, electron rich) to 100 kcal/mol (blue, electron poor).

X-ray and NMR structures of a wide range of reader proteins bound to methylated Lys provide a general picture into how selectivity is achieved for different methylation states of Lys. The structural aspects of these proteins have been reviewed in detail elsewhere.6,20,45–48 Herein we provide a brief summary of key examples that probe the mechanism of recognition. Protein binding pockets containing 2-4 Tyr, Trp, and/or Phe selectively bind to the methylammonium of KMe3 through cation-π interactions with the N-CH3 groups. Selectivity between different methylation states of Lys is achieved by tuning the ratio of aromatic residues versus acidic residues, which can form salt bridges (electrostatic and hydrogen bonding interactions) with the lower methylation states of Lys.6,46 Additionally, selectivity for lower methylation states over


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KMe3 is typically provided by the steric occlusion of larger methylated ammoniums.49 For example, human lethal(3) malignant brain tumor repeat-like protein-1 (L3MBTL1/2) recognizes H4 K20Me2 (Kd = 11 μM) and H2 K20Me1 (Kd = 14 μM) with greater than 10-fold selectivity over unmethylated Lys and KMe3. Binding occurs via interaction of the methylammonium with a binding pocket consisting of three aromatic residues and one Asp, which contributes a negative charge.6,50,51 Crystal structures confirm that binding occurs exclusively through insertion of KMe2 or KMe into a deep cavity, forming a direct hydrogen bond between the methylammonium and Asp in addition to complementary cation-π interactions with the Trp, Tyr, and Phe. Conversely, binding to KMe3 is prevented by steric occlusion from the aromatic cavity.51 Several studies have investigated the contribution of electrostatic interactions and/or salt bridges to binding and selectivity of reader proteins. In the case of the HP1 chromodomain, which has a shallow binding pocket (Fig. 2), E52 forms a water-mediated salt bridge with KMe2 and a water-mediated electrostatic interaction with KMe3, and exhibits minimal selectivity (~1.5-fold) for KMe3 over KMe2. The E52Q mutation maintains hydrogen bonding but removes the electrostatic interaction. Interestingly, this has no effect on binding to KMe3, whereas the binding to KMe2 is reduced 2.6-fold (ΔΔG = 0.6 kcal/mol), resulting in an improved selectivity for KMe3 of 3.4-fold.

A

tion of different reader proteins.

A

B

Figure 3. A. Aromatic cage of BPTF PHD domain (green) formed by three Try and on Trp bound to KMe3 (yellow) (2FUU). B. Aromatic cage of the Y17E mutant of BPTF PHD domain (green) bound to KMe2 (yellow) (pdb: 2RI7). The hydrogen bond between Y17E and KMe2 is shown with the yellow dashed line.

Protein Recognition of Methylated Arg Like protein-Lys methylation, protein-Arg methylation is a common PTM, which has received widespread attention in recent years. It is involved in the regulation of gene transcription, RNA processing, signal transduction, DNA repair, and histone crosstalk.54,55 Arg contains five potential hydrogen bond donors which can be used to stabilize interactions with DNA, RNA and proteins.55 Arg can be methylated up to two times to form either mono(RMe), symmetric di- (sRMe2), or asymmetric dimethylarginine (aRMe2) (Fig. 4). Arg is methylated by protein arginine methyltransferases (PRMTs), which can produce either aRMe2 (PRMTs 1, 2, 3, 4, 6 and 8) or sRMe2 (PRMTs 5 and 7).4

A

B

Figure 2. A. Aromatic cage of HP1 chromodomain (green) formed by two Tyr and one Trp bound to KMe3 (yellow) exhibiting a wider aromatic cage and neighboring Glu that con52 tributes to KMe2 binding, but not KMe3 binding (pdb: 1KNE); B. Aromatic cage of L3MBTL1 (green) bound to KMe (yellow) exhibiting a tighter aromatic cage and direct salt bridge to Asp (pdb: 2RHY).

In the BPTF PHD finger domain (Fig. 3), mutation of Y17 to E reversed the selectivity for KMe3 relative to KMe2.50 This mutation introduced a salt bridge in the binding pocket, resulting in a 2:1 preference for KMe2 over KMe3, rather than the 3:1 preference for KMe3 in the wt protein. Interestingly, in this protein, the Y17Q mutant resulted in weaker binding to both KMe2 and KMe3, with a larger effect on KMe3 binding. A similar result was observed for the Sfg29 tandem tudor domain bound to H3 K4Me2/3.53 This is the opposite of the observation in the HP1 chromodomain. Thus, it appears that the contribution of salt bridges and electrostatic interactions is dependent on the nature of the binding pocket. This may provide a valuable approach to achieving selective inhibi-

B Figure 4. A. Arg methylation states. B. Electrostatic Potential Maps of Arg sidechain methylation states corresponding to those in A. Electrostatic potential maps were generated with MacSpartan: HF/6-21g; isodensity value = 0.02; range = 100 (red, electron rich) to 200 kcal/mol (blue, electron poor).

Similar to Lys methylation, the methylation of Arg does not affect the overall charge of the residue; however, the addition of methyl groups decreases the hydrogen bonding capacity, weakening its solvation.56 Upon methylation, the size and hydrophobicity of the guanidinium are increased, also increasing steric bulk of the residue and resulting in dispersion of the positive charge to the pendant methyl groups (Fig. 4).57



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Consequently, the methyl groups possess a δ+ charge, making good contacts for CH3(δ+)-π interactions in aromatic protein binding pockets.7 Each of the four known crystal or NMR structures of Tudor domains bound to aRMe2 or sRMe2 bind the methylated guanidinium in an aromatic pocket with affinities ranging from 5 μM to > 200 μM.58–60 The binding selectivity occurs via a combination of CH-π interactions with the N-CH3(δ+) groups, cation-π and π-π stacking of the guanidinium group with two aromatic rings, NH-π interactions, and hydrogen bonds (Fig. 5).59–61 Selectivity for Arg over KMe3 is achieved because the binding pockets are narrow, limiting binding by KMe3 through steric interactions.45

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cation-π interaction that drives recognition of cationic guests by cyclophanes, and hydrophobicity only moderately influences binding affinity. Analogous to experiments with small molecules, Waters demonstrated that a reader protein, the HP1 chromodomain also relies on the cation-π interaction to bind to KMe3 more favorably than its all-carbon analog, as described above.41

Figure 6. Dougherty’s Cyclophane DC1 and derivatives DC2 and DC3 used to study intermolecular cation-π and in water, 38,62–66 and examples of guests for binding studies.

A

B

Figure 5. eTud11 binding to sRMe2 in an aromatic cage consisting of 3 Phe and one Tyr, which provide cation-π interactions and π-π stacking, and an Asn that provides a hydrogen 61 bond (pdb: 3NTH). B. Recognition of aRMe2 by TDRD3 in an aromatic binding pocket with one Phe and 3 Tyr residues through cation-π interactions and π-π stacking (pdb: 58 2LTO).

Binding of Cationic Guests with Synthetic Hosts While studies of protein recognition of methylated Lys and Arg have provided some mechanistic insight into these binding events, model systems have provided the preponderance of information. Early computational studies on gas phase binding energies revealed that Na+ (ΔH° = -28 kcal/mol), Li+ (ΔH° = -38 kcal/mol), and even NH4+ and Me4N+ interact favorably with benzene in the gas phase.30 In an effort to translate these gas phase affinities to solution-based binding constants, Dougherty generated a suite of cyclophanes (aromatic macrocycles) to systematically probe binding interactions between aromatic macrocycles and a series of cationic, neutral, hydrophobic, and hydrophilic guests (Fig. 6).38,62–66 In a series of papers, he compared the binding constants of nearly isostructural compounds that varied in hydrophobicity, amine placement, or charge (Fig. 6). These studies demonstrated that cation-π interactions with quaternary ammonium salts out-compete hydrophobic interactions with uncharged groups of the same size and shape, despite the large difference in desolvation costs. Further evidence was given through NMR studies, which revealed that the aromatic pocket preferentially interacts with the cationic ammonium group of G3 in water over the hydrophobic tert-butyl group, despite the ammonium being better solvated in water. If hydrophobicity were the major driving force for binding, the more hydrophobic tertbutyl group would be bound more tightly than the ammonium, however, this is not the case. Therefore, it is the

In addition to the cation-π interaction, Dougherty and coworkers explored the influence of carboxylates proximal to the aromatic pocket of the cyclophanes.66 DC1 does not bind primary ammonium ions due to the greater cost of desolvation of the well-hydrated cation in water. Interestingly, placement of a carboxylate in close proximity to the binding pocket improves binding through electrostatic and/or hydrogen bonding interactions. Binding studies with diamines revealed that this charge-pairing was distance-dependent, suggesting that electrostatic interactions between ammoniums and carboxylates rely on optimal substrate positioning and are not the result of longrange electrostatic interactions. Additionally, the proximal carboxylates only slightly improved binding affinity to quaternary alkyl ammoniums in water (-0.1-0.3 kcal/mole for DC2 and -0.13-0.7 kcal/mole for DC3, which contains four additional carboxylates), which can likely be attributed to the diffuse nature of the cationic charge in quaternary ammoniums.66 Together these studies establish the cation-π interaction as a substantial driving force in molecular recognition in water that serves as the foundational driving force for binding in the generation of small molecule receptors for both methylated Lys and Arg. Recognition of Methylated Lys While the work of Dougherty and others30,39 defined the nature of cation-π interactions with quaternary ammonium salts, these host systems did not lend themselves to investigation of the influence of Lys or Arg methylation on the magnitude and driving force of a cation-π interaction, due to the macrocyclic nature of the receptors. To address this, Waters and co-workers utilized a betahairpin peptide model system to study the interaction with methylated Lys and Arg with a single aromatic residue. In these studies, they found that methylation of Lys or Arg increases the magnitude of the cation-π interaction 2-3 fold, with the methyl groups forming direct contact with the aromatic ring. For KMe3, the magnitude of the interaction amounts to 1 kcal/mole for a single cation-π interaction. Moreover, the charge was found to be critical for the interaction even with a single aromatic residue, similar to observations in Dougherty’s cyclophane and in


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the HP1 chromodomain. Design of small molecule receptors. The binding pockets of proteins and the interactions that drive substrate recognition have inspired the design of small molecule receptors for methylated Lys and Arg. The most successful receptors for methylated Lys mimic the aromatic pockets of reader proteins by combining aromatic residues to provide strong cation-π interactions and anionic functionality for electrostatic and hydrogen-bonding interactions. In the following section, we highlight a few of the most successful classes of small molecule receptors for methylated Lys. A variety of small molecule receptors for KMe3 has been synthesized in recent years, including calix[4]arenes,67,68 cucurbit[6/7]urils,69 and mercaptophanes70 (Fig. 7). These receptors bind to KMe3 with selectivity over the lower methylation states of Lys, and in some cases bind KMe3 with affinity and selectivity comparable to native reader proteins under mild salt conditions (10 to 40 mM buffer). In each of the receptors, KMe3 binding is driven by cation-π and hydrophobic interactions with the N-CH3 group and the hydrocarbon side chain. Selectivity for KMe3 over unmethylated Lys is driven by a combination of favorable cation-π interactions with KMe3 and the high cost of desolvation of unmethylated Lys in water.

Figure 7. Receptors that selectively recognize KMe3: Sul71–74 67 fonatocalix[4]arene (CX4), CX4-Ph, and cucurbit[7]uril 69 (CB7).

Sulfonatocalix[4]arene. Sulfonatocalix[4]arene (CX4) is a macrocyclic molecular receptor with an aromatic interior that exists in a cup-like conformation with a narrow lower rim and a wide upper rim, which is decorated with anionic sulfonates (Fig. 7). The sulfonates provide both water solubility and an ability to interact with a variety of positively charged guests including the amino acid form of Lys (Kd = 1.9 mM) in water.68 Hof repurposed CX4 to study its interaction with histone PTMs.71,72 NMR binding studies revealed that CX4 binds to the amino acid form of KMe3 (Kd = 27 μM) with 60-fold improved binding affinity over Lys through a change in binding mechanism. When binding to Lys, the entire side chain spans the sulfonated rim of CX4, forming electrostatic interactions between the sulfonates and N-terminal and side chain ammoniums, and weak hydrophobic interactions between the aliphatic Lys side chain and hydrophobic interior of the macrocycle.74 In contrast, the trimethylammonium of KMe3 interacts with both the aromatic core of the binding pocket and the sulfonates, via a combination of cation-π interactions and electrostatic interactions.67,75 Similar to proteins with shallow binding pockets, like the HP1 chromodomain, CX4 binds KMe3 with 3-fold selectivity over KMe2 (Kd = 95 μM), possibly due to the decreased cation-π interactions and/or the increased cost of desolv-

ation of KMe2. Not surprisingly, elimination of the positive charge of Lys via acetylation diminishes binding.74 Binding affinity is improved in the context of a short histone H3 peptide tetramer, Ac-RK(Men)ST-NH2 (n = 0 or 3) for both KMe3 (Kd = 10.3 μM) and Lys-containing peptides (Kd = 180 μM). This improved binding affinity and decrease in selectivity can be attributed to additional non-specific electrostatic interactions provided by the neighboring Arg in the peptide sequence and a potential disruption of the binding modes of the free amino acids. In an effort to improve KMe3 binding, Hof synthesized a series of derivatives of CX4, leading to the discovery of CX4-Ph, a trisulfonated calix[4]arene functionalized with an additional phenyl ring to establish a deeper aromatic pocket capable of more interactions with the KMe3 side chain (Fig. 7).67 1H NMR revealed that the deeper binding pocket increased contacts with the aliphatic KMe3 side chain, improving binding by a factor of 2.5 over CX4 (Kd = 13-16 μM), but more importantly, improving the selectivity over unmethylated Lys to 150-fold (Kd = 2.4 mM), likely because of the increased hydrophobicity of the binding pocket and the loss of a negatively charged sulfonate on the upper rim of the macrocycle.67 Multivalent CX4-Ph Receptors. Recently, CX4-Ph was conjugated to short peptides through a p-carboxylate on the pendant phenyl ring to generate mono-, di-, and trivalent CX4-Ph binding motifs.76 The attachment of two, or three CX4-Ph units improved binding affinity for an H3 K27Me3 peptide from the monovalent derivative (Kd = 8.9 μM) resulting in tight binding (Kd = 0.39 μM) for both receptors. Selectivity over the unmethylated peptide was moderately increased for the divalent receptor (from 2fold to 4-fold selectivity), but actually decreased for the trivalent receptor (1.3-fold) with relatively tight binding to the unmethylated peptide (Kd = 1.8 μM and 0.51 μM for the di- and trivalent receptors, respectively). Arg and Lys both strongly influenced binding affinity to H3 and H4 peptide guests, and the minimal improvement in selectivity suggests that the non-specific electrostatic interactions between the positively charged peptide overpower the more selective cation-π interactions that drive selectivity for KMe3 over Lys. Cucurbit[7]uril. Cucurbit[n]urils (CB[n]) are macrocycles comprised of [n] bridged glycoluril subunits (Fig. 7). CB[n]’s are neutral and do not contain aromatic surfaces, but rely on binding through the hydrophobic interior and ion-dipole interactions with the carbonyl rims of the. CB7 binds KMe3 the amino acid version of KMe3 (Kd = 0.53 μM) with 3500-fold selectivity over the unmethylated Lys (Kd = 1.9 mM) through a combination of the hydrophobic effect and van der Waals interactions on the interior of the macrocycle, along with ion-dipole interactions with the polar carbonyl head groups and the free ammonium.69 CB7 is the most selective synthetic macrocycle reported for KMe3 as an amino acid over unmethylated Lys; however, binding studies with peptide, or protein variants of KMe3 have not been reported to the best of our knowledge. Given the significant selectivity of CBs for dications, which allows for favorable ion-dipole interac-



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tions at both rims, it is expected that binding will be significantly weaker to KMe3 in the context of a peptide. Mercaptophanes. The Waters group has also developed a number of small molecule receptors with proteinlike affinity and selectivity for the various methylation states of Lys. Motivated by work by Otto and Sanders developing dynamic combinatorial chemistry (DCC) to develop synthetic receptors for a wide range of cations in aqueous solution, we used DCC77 with disulfide exchange in water to rapidly screen a large number of potential receptors for a guest of interest, including methylated Lys and Arg. We typically use water-soluble aromatic compounds with two thiols to generate aromatic macrocycles with negatively charged exteriors, aromatic interiors, and disulfide linkages between monomer subunits. The first receptor isolated in our group using DCC was A2B (both rac- and meso-), which exhibited binding affinity for KMe3 (Kd = 2.6 μM) and selectivity over KMe2 (Kd = 6.3 μM) and Lys (Kd = 22 μM) that was comparable to the HP1 chromodomain.70,78 The negatively charged exterior aids in binding to histone H3 peptides that contain a neighboring Arg, while selectivity for KMe3 is attributed to the increased cation-π interactions afforded by the additional N-CH3 group and the increased cost of desolvation for the lower methylation states of Lys. Using DCC to systematically vary the binding pocket, we explored the influence of non-covalent interactions, pocket size, and macrocycle shape on molecular recognition of methylated Lys in water. Preliminary library studies revealed the importance of the A2-moiety, which was conserved in macrocycles that bound KMe3, likely through insertion of N-CH3 groups into the A2- cleft. This observation led us to synthesize a series of receptors by pairing monomers C-N with A2 (Fig. 8).79

70

Figure 8. Redesign of A2B to explore the effects of electrostatic interactions and a deeper aromatic pocket in the 78,79 A2X framework.

Monomer C was designed to probe the influence of charge positioning. Moving the carboxylate from the meta- to the ortho- position improved binding to KMe2 (Kd = 2.8 μM), but did not affect binding to the other methyl-

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ation states of Lys. This suggests that binding to KMe2 is improved through the formation of a hydrogen bond between the carboxylate of C and KMe2 within the binding pocket, similar to interactions in the binding pockets of reader proteins.6,48 When charge near the binding pocket was increased in A2E, binding affinity was improved by ~ 10-fold for all methylation states of Lys (Kd for KMe3 = 0.191 μM); but selectivity was not influenced, suggesting that electrostatic interactions near the binding pocket improve binding affinity, but do not discriminate between methylation states of Lys. Binding pocket depth had the greatest influence on target selectivity. The benzene derivatives, B, C, and E produce relatively shallow binding pockets, similar to the surface exposed pockets of the HP1 chromodomain and the PHD finger of human BPTF. Likewise, these receptors exhibit ~ 2-fold selectivity for KMe3 over KMe2. In contrast to the benzene-derived monomers, molecular modeling suggests that A2G can adopt a deeper cavity when bound to KMe3, increasing the contacts along the KMe3 side chain. Binding studies revealed that binding affinity for KMe3 was only improved by ~ 2-fold (Kd = 1.4 μM); however, selectivity over KMe2 (Kd = 13.2 μM) was improved to 9.5-fold with 40-fold selectivity over Lys (Kd > 58 μM). Similarly, A2N possesses a deepened hydrophobic pocket and increased cation-π interactions with alkyl ammonium guests, improving binding of KMe3 by ~ 9fold (Kd = 0.30 μM).78 Extensive investigation into the mechanisms of KMe3 binding revealed that the N-CH3’s are buried within the aromatic pocket, while the N subunit of A2N forms strong contacts with the aliphatic Lys side chain, similar to CX4-11. Selectivity over the lower methylation states was also improved, with 13.5-fold selectivity for KMe3 over KMe2 (Kd = 4.1 μM) and 35-fold selectivity over Lys (Kd = 10.5 μM). Interestingly, selectivity over Lys was sequence dependent and could be increased to over 200-fold by removing neighboring Arg residues, which play a role in mediating receptor binding, potentially through stacked salt bridges.80 A2H was synthesized as a naphthalene-derived surrogate for monomer N. Interestingly, DCC libraries revealed a self-templating effect in which two isomers of A2H dominated library composition as the most thermodynamically stable species in the library. Molecular modeling showed the unsubstituted portion of the naphthalene fit ideally into the A2-cleft of A2H, and binding studies revealed only weak binding to KMe3 and little to no selectivity over Lys, demonstrating that the intermolecular forces for binding could not overcome the intramolecular interactions between the H-naphthalene and the A2-cleft. Other Lys binders. Two additional classes of receptors that exhibit interesting molecular recognition properties and provide insight into binding mechanisms include carboxylatopillar[5]arenes (CP5A)81 and a unique class of indole receptors82,83 (Fig. 9). Pillararenes make up a novel class of supramolecular hosts that can recognize biologically relevant targets in water. In 2013, Li and coworkers81 reported a carboxyla-


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topillar[5]arene (CP5A) capable of binding to basic amino acids Arg, His, Lys and KMe3 with high micromolar affinity. Binding occurred via electrostatic interactions with the carboxylates and the cationic guest as well as hydrophobic interactions between the aromatic core of CP5A and the aliphatic side chain of the amino acid. Interestingly, CP5A selectively binds to unmethylated Lys over KMe3 likely due to the loss of hydrogen bond donors in KMe3. Similar to proteins with deep, but narrow binding cavities, such as L3MBTL1/2, KMe3 is too large to fit inside the binding pocket and is excluded from the aromatic core of the macrocycle through steric interactions. The carboxylates on both sides of the macrocycle contribute to Lys binding by forming electrostatic interactions with both the ε-NH2 and the zwitterionic motif of the amino acid.

Figure 9. Small molecule receptors that do not bind KMe3 either because of steric occlusion (CP5A)81 or from receptor flexibility (Trp(Bn)-Trp(Bn) and CICR).82,83

In contrast to the receptors discussed to this point, Hof’s indole receptors were designed to be highly flexible and benefit from an induced fit when binding to methylated Lys. Two sets of indole-containing receptors were used to probe the influence of the electron-rich aromatic indole on binding to alkylammoniums in water.82,83 Trp is abundant in reader protein binding pockets, and many crystal structures show direct interactions between bound KMe3 and the indole of Trp. Despite molecular modeling that suggested favorable binding pocket formation, the Trp(Bn)-Trp(Bn) receptors were only moderate binders of alkylammonium guests (Kd = 70 mM for AcCH).82 A slightly more rigid receptor, CICR exhibited moderately increased affinity for KMe3, however, binding was still relatively weak (Kd = 4.0 mM)83 when compared to macrocyclic receptors. While carboxylate position did contribute to binding affinity, the receptors remained too hydrophobic and collapsed in solution, unable to bind guests; or too flexible and disordered in solution, requiring significant conformational restraint in order to bind. This study revealed the importance of the rigidity of the binding pocket for tight binding, and confirmed the importance of charge positioning in improving binding interactions. Molecular Recognition of Methylated Arg The guanidinium group of Arg and methylated Arg provides the opportunity to form cation-π interactions with greater surface area and additional possibility of a ππ stacking interaction. Moreover, while the edge of a guanidinium group hydrogen bonds with water, the faces are not well solvated, increasing the favorability of the cation-π interaction.57 To gain insight into the effect of methylation on cation-π interactions involving Arg, the Waters group studied the interaction of aRMe2 and sRMe2 with Trp in a beta-hairpin peptide, similar to the

studies of KMe3-Trp interaction in a beta-hairpin described above. They found that methylation of Arg doubles the strength of the interaction from 0.5 to 1 kcal/mol, and the N-CH3 groups interact directly with the face of the Trp residue. Synthetic Receptors for Arg and Methylated Arg. The research on receptors for Arg and methylated Arg lag significantly behind that of methylated Lys. Early work focused on binding of the parent amino acid Arg, resulting in several synthetic receptors that possess hydrophobic interiors with negatively charged exteriors.66,72,73,84,85 While contributions from these interactions vary in each receptor, the primary binding forces governing recognition of methylated Arg are cation-π, ππ, hydrophobic, and electrostatic interactions with carboxylates appended from the aromatic core. Molecular Tweezers. A wide range of molecular tweezers, a class of preorganized, rigid U-shaped receptors with high electron density and hydrophobicity on their inner surface, has been reported.86 In 2005, Klarner described a phosphate-modified, water soluble molecular tweezer that binds both Lys (Kd ≈ 40 μM) and Arg (Kd ≈ 130 μM) as protected amino acids (to minimize additional interactions with the zwitterionic backbone).84 Cation-π interactions and dispersion forces along the amino acid side chain are the primary driving forces for binding. These receptors have not been explored for methylated Lys or Arg. Sulfonatocalix[n]arenes. A range of sulfonatocalix[n]arenes display relatively weak binding affinity for the basic amino acid of Arg (Kd ≈ 0.6 - 3.0 mM).72 Binding likely occurs through electrostatic and hydrogen bonding interactions between the guanidinium and sulfonates, with addition CH-π interactions between the Arg side chain and the aromatic core of the calix[n]arene. Dougherty’s Cyclophanes. While DC1 does not bind unmethylated Lys or Arg, which are well solvated and have a high cost of desolvation in water, installation of carboxylates on the rim of the macrocycle to produce DC2 and DC3 (Fig. 6)66 introduces a distance-dependent charge-pairing effect and improves binding affinity of Arg (Kd ≈ 0.1 – 1.0 mM) and Lys by up to 1.0 kcal/mol. Binding to Arg is 1.0 kcal/mol more favorable than Lys, likely due to improved cation-π and π-π stacking interactions with its delocalized π system and flat surface, in addition to its relative hydrophobicity along its π-surface.87 Cucurbit[7]uril. In addition to KMe3, CB7 also binds to the amino acid forms of aRMe2 and sRMe2.69 Interestingly, neutral cucurbit[n]urils, which lack aromaticity, bind methylated Arg through a combination of hydrophobic and van der Waals interactions on the interior of the macrocycle, and ion-dipole interactions with the carbonyl rims. CB7 binds sRMe2 (Kd = 160 μM) over aRMe2 (Kd = 500 μM) and exhibits considerable selectivity over RMe (56-fold) and Arg (32-fold), likely due to their higher cost of desolvation. 1H NMR revealed that binding of the methylated guanidinium occurs within the host cavity, but is still complemented by strong ion-dipole interactions between the free ammonium of the amino acid and



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the CB7 carbonyl groups. Loss of the ammonium group by incorporation of aRMe2 and sRMe2 into peptides may significantly weaken binding, although this was not reported. Carboxylatopillar[5]arene. The carboxylatopillar[5]arene exhibits moderate affinity for the Arg amino acid (Kd = 170 μM) in water, which is likely due to the multiple hydrogen bonds the guanidinium can make with the carboxylates, and shows little decrease in affinity in the context of a peptide consisting of Ala-Arg-Ala (Kd = 240 μM). Mercaptophanes. In a continuation of the iterative redesign of the KMe3 receptor, A2B, the Waters group identified a new receptor using DCC, A2D, that exhibits preferential binding of aRMe2 with a Kd of 5 μM with greater than 7-fold selectivity over sRMe2 and more than 10-fold selectivity over the unmethylated peptide (Figure 8).88 These affinities and selectivities, which are derived primarily from interaction with the methylated side chain, are comparable to those exhibited by native methyl Arg binding domains, which also recognize the surrounding residues and protein backbone in addition to the side chain.59–61,89 This receptor provides an increase the size of the binding pocket and provides a large π-surface to improve πstacking and cation-π interactions. While the magnitude of a cation-π interactions has been shown to be equivalent for the two isomers of RMe2,57 NMR studies indicate that aRMe2 fits both methyl groups into the binding pocket whereas the two methyl groups of sRMe2 are sterically occluded. Surprisingly, the binding affinity of A2D for H3 K9Me3 was found to be equivalent to that of H3 R8aMe2, showing no distinction between the two modifications and pointing to challenges in molecular distinction between aRMe2 and KMe3 with macrocyclic hosts. Presumably more fine-tuning of the cavity size and shape is needed to exclude the spherical NMe3 in favor of the flat methylguanidinium.

Conversely, selective recognition of methylated Arg has proven to be challenging. Proteins recognize RMe2 residues in surface-exposed aromatic cages that are slightly narrower than KMe3 binding pockets. Cation-π, π-π stacking, van der Waals, and hydrophobic interactions drive selectivity for methyl-Arg, yet only a few receptors that selectively bind methylated Arg have been discovered. The disproportion between Lys and Arg receptors could be due to the myriad non-covalent contributions that influence binding of aRMe2 and sRMe2, such as hydrogen bonding, NH-π interactions, and stacking, significantly complicating selective binding of RMe2 relative to the spherical KMe3. Nonetheless, Arg-receptors currently are being studied in detail, and we expect new, selective motifs will arise in the near future. The development and characterization of protein binding interactions and small molecule receptors continues to enhance our understanding of molecular recognition in water. Recently, CX4 was used with a solvatochromatic dye in an indicator displacement assay (IDA)90 to monitor real-time, enzymatic Lys methylation.91 IDAs have been used to create sensor arrays for several receptors to read out the presence of different methylation states of Lys in histone peptides and to disrupt interactions between KMe3 and native reader proteins.92–94 As our knowledge of molecular recognition of histone PTMs progresses, the ongoing development of small molecule receptors, and emphasis on their non-covalent interactions with high value targets like KMe3 and RMe2 will drive the evolution of novel protein inhibitors, new anticancer therapeutics, and expand upon current enzyme assays and sensor arrays.

Conclusions and Outlook. Recent investigations into the elucidation of small molecule binding of methylated Lys and Arg have provided considerable insight into the non-covalent interactions that drive molecular recognition in water. In both reader proteins and small molecule receptors, binding of KMe3 is primarily driven by the cation-π interactions between NCH3(δ+) and the negatively charged π-surface of aromatic rings. Selectivity for KMe3 over lower methylation states is derived from a combination of increased cation-π interactions with higher methylation states, and the increased cost of desolvation as the number of methyl groups is decreased. Selectivity for lower methylation states of Lys is imparted in both proteins and receptors through steric occlusion of KMe3 in narrow binding pockets coupled with favorable electrostatic interactions. A number of small molecule receptors capitalize on these characteristic binding pocket attributes, and rival or surpass the binding affinities and selectivities of native reader proteins, despite the large disparity in size.

† Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, United States

AUTHOR INFORMATION Corresponding Authors *Joshua E. Beaver, email: [email protected] *Marcey L. Waters, email: [email protected]

Present Addresses

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources W. M. Keck Foundation (MLW), NSF CHE-1306977 (MLW), and NSF DGE-1144081 (JEB) and Keck foundation

ACKNOWLEDGMENT We gratefully acknowledge funding from the W. M. Keck foundation. This material is based in part upon work supported by the National Science Foundation under grant no. CHE-1306977, and the National Science Foundation Gradu-


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ate Research Fellowship to J.E.B under grant no. DGE1144081.

ABBREVIATIONS PTM, post-translational modification; Lys, lysine; Arg, arginine; Trp, tryptophan; Phe, phenylalanine; Tyr, tyrosine; Asp, asparagine; Ala, alanine; KMe3, trimethyllysine; KMe2, dimethyllysine; RMe, monomethylarginine; aRMe2, asymmetric dimethylarginine; sRMe2, symmetric dimethylarginine; PRMT, protein arginine methyltransferase; HP1, Drosophila melanogaster heterochromatin protein 1; eTud11, Drosophila melanogaster TUD protein; BPTF, bromodomain PHD finger transcription factor; PHD, plant homeodomain; TDRD3, tudor domain containing protein 3; H1-4, histone proteins; L3MBTL, lethal(3)malignant brain tumor-like protein; Sfg29, SAGA-associated factor 29; DC, Dougherty’s cyclophane; CX4, sulfonatocalix[4]arene; CX4-Ph, phenyl sulfonatocalix[4]arene; CB7, cucurbit[7]uril; A2X (A2B, A2C, A2D, A2E, A2G, A2H, A2N): mercaptophane receptors shown in Figure 8; CP5A, carboxylatopillar[5]arene; CICR, carboxyindole-containing receptor; HF/6-31G*, Hartfree-Fock Theory; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; IDA, indicator displacement assay.

Key Words Epigenetics: Heritable modifications of gene expression and phenotype and that occur without alteration of the genotype. PTM (Posttranslational Modification): Covalent modification of a protein following protein synthesis. Common PTMs include acetylation, phosphorylation, methylation, ubiquitination, and glycosylation. Molecular Recognition: The combination of noncovalent interactions between two molecules that results in a selective noncovalent binding interaction. Reader Protein: Proteins that recognize PTMs on the Nterminal tails of histones and recruit proteins and enzymes to the nucleosome to regulate gene transcription. Histone Code: The network of PTMs on the N-terminal histone tails that recruit reader proteins to the nucleosome. Lys and Arg Methylation: Lys and Arg methylation is intimately involved in the regulation of gene transcription. Lys can be methylated to form mono- (KMe), di(KMe2) and trimethyllysine (KMe3), and Arg can be methylated to mono- (RMe), asymmetric di- (aRMe2), and symmetric dimethylarginine (sRMe2). Cation-π π Interaction: The coloumbic attraction between a cation and the partially negatively charged π-surface of an aromatic ring, which is arises from the quadrupole moment of the ring. This coloumbic attraction is the primary binding force for recognition of quaternary ammoniums, such as trimethyllysine, by reader proteins in water. Synthetic Receptor: A synthetic molecule, usually < 1-2 kDa, that binds to a target of interest through a combination of non-covalent interactions such as cationπ hydrophobic interactions.

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KMe3 Reader Protein

KMe3 Receptor

Binding Mechanism


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Molecular Recognition of Lys and Arg Methylation - American

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