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

   https://doi.org/10.1002/pro.4533  

   Travis, Christopher R.; Francis, Denver Y.; Williams, Jr., David C.; Waters, Marcey L. (December 2022, Protein Science)

   Abstract 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. 

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2. Proline-Aromatic Sequences Stabilize Turns via C–H/π interactions in both cis-Proline and trans-Proline

   https://doi.org/10.26434/chemrxiv-2023-7nb44  

   Ganguly, Himal K; Elbaum, Michael B; Zondlo, Neal J (July 2023, chemRxiv)

   In proteins, proline-aromatic sequences exhibit increased frequencies of cis-proline amide bonds, via proposed C–H/π interactions between the aromatic ring and either the proline ring or the backbone C–Hα of the residue prior to proline. These interactions would be expected to result in tryptophan, as the most electron-rich aromatic residue, exhibiting the highest frequency of cis-proline. However, prior results from bioinformatics studies on proteins and experiments on proline-aromatic sequences in peptides have not revealed a clear correlation between the properties of the aromatic ring and the population of cis-proline. An investigation of the effects of aromatic residue (aromatic ring properties) on the conformation of proline-aromatic sequences was conducted using three distinct approaches: (1) NMR spectroscopy in model peptides of the sequence Ac-TGPAr-NH2 (Ar = encoded and unnatural aromatic amino acids); (2) bioinformatics analysis of structures in proline-aromatic sequences in the PDB; and (3) computational investigation using DFT and MP2 methods on models of proline-aromatic sequences and interactions. C–H/π and hydrophobic interactions were observed to stabilize local structures in both the trans-proline and cis-proline conformations, with both proline amide conformations exhibiting C–H/π interactions between the aromatic ring and Hα of the residue prior to proline (Hα-trans-Pro-aromatic and Hα-cis-Pro-aromatic interactions) and/or with the proline ring (trans-ProH-aromatic and cis-ProH-aromatic interactions). These C–H/π interactions were strongest with tryptophan (Trp) and weakest with cationic histidine (HisH+). Aromatic interactions with histidine were modulated in strength by His ionization state. Proline-aromatic sequences were associated with specific conformational poses, including type I and type VI β-turns. C–H/π interactions at the pre-proline Hα, which were stronger than interactions at Pro, stabilize normally less favorable conformations, including the ζ or αL conformations at the pre-proline residue, cis-proline, and/or the g+ χ1 rotamer or αL conformation at the aromatic residue. These results indicate that proline-aromatic sequences, especially Pro-Trp sequences, are loci to nucleate turns, helices, loops, and other local structures in proteins. These results also suggest that mutations that introduce proline-aromatic sequences, such as the R406W mutation that is associated with protein misfolding and aggregation in the microtubule-binding protein tau, might result in substantial induced structure, particularly in intrinsically disordered regions of proteins. 

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3. Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

   https://doi.org/10.3390/biom10020273  

   Afrose, Fahmida; Koeppe II, Roger E. (February 2020, Biomolecules)

   Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues. 

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4. Cavity-containing aromatic oligoamide foldamers and macrocycles: progress and future perspectives

   https://doi.org/10.1039/D2OB01467J  

   Sobiech, Thomas A.; Zhong, Yulong; Gong, Bing (September 2022, Organic & Biomolecular Chemistry)

   As a major class of foldamers, aromatic oligoamide foldamers have attracted intense interest. The rigidity of aromatic residues and amide linkages allows the development of foldamers with readily predictable, stable conformations. Aromatic oligoamide foldamers having backbones fully constrained by intramolecular hydrogen bonds have attracted wide attention. Depending on their lengths, such foldamers adopt crescent or helical conformations with highly negative inner cavities. Cyclizing the backbone of the aromatic oligoamides affords the corresponding macrocycles which are characterised by persistent shapes and non-deformable inner cavities. With their defined, inner cavities, such aromatic oligoamide foldamers and macrocycles have served as hosts for cationic and polar guests, and as transmembrane channels for transporting ions and molecules. Recent synthetic progress resulted in the construction of multi-turn hollow helices that offer three-dimensional inner pores with adjustable depth. Reducing the number of backbone-constraining hydrogen bonds leads to oligoamides which, with their partially constrained backbones, undergo either solvent- or guest-dependent folding. One class of such aromatic olgioamide foldamders, which offer multiple backbone amide NH groups as hydrogen-bond donors, are designed to bind anions with adjustable affinities. 

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5. Serine-404 Phosphorylation and the R406W Modification in Tau Stabilize the cis-Proline Amide Bond, via Phosphoserine-Proline C–H/O and Proline-Aromatic C–H/π Interactions

   https://doi.org/10.26434/chemrxiv-2023-w152b  

   Ganguly, Himal K; Elbaum, Michael B; Zondlo, Neal J (July 2023, chemRxiv)

   Tau misfolding, oligomerization, and aggregation are central to the pathology of Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), frontotemporal dementia, and other tauopathies. Increased phosphorylation of tau is associated with conformational changes that are not fully understood. Moreover, tau oligomerization and aggregation are associated with proline cis-trans isomerism, with the phosphorylation-dependent prolyl isomerase Pin1 reducing tau hyperphosphorylation and aggregation. The FTDP-17 tau mutation R406W is frequently used in animal models of Alzheimer's disease, due to earlier onset of the AD phenotype. Despite its extensive application, the mechanisms by which tau-R406W leads to enhanced aggregation and neurotoxicity are poorly understood. Peptides derived from the tau C-terminal domain were examined by NMR spectroscopy as a function of residue 406 identity (Arg versus Trp) and Ser404 phosphorylation state. The R406W modification led to an increased population of Pro405 cis amide bond, which is stabilized by cis-proline-aromatic C–H/π interactions. Ser404 phosphorylation also resulted in an increase in cis amide bond, via a proposed C–H/O interaction between the Pro Hα and the phosphate that stabilizes the cis conformation. An analogous C–H/O interaction was observed in Glu-cis-Pro sequences in the PDB, and is proposed to be the basis of the increased propensity for cis amide bonds in Glu-Pro sequences. The higher activation barriers for proline cis-trans isomerization observed at pSer-Pro and pThr-Pro sequences are proposed to be due to both (a) an intraresidue phosphate-amide bond that stabilizes the trans-proline conformation and (b) the cis-stabilizing proline-phosphate C–H/O interaction identified herein. The combination of both pSer404 and R406W resulted in a further increase in the population of cis amide bond. In contrast to expectations, the R406W modification led to increased dephosphorylation of either pSer404 or pSer409 by PP2A, and had no effect on phosphorylation of Ser404 by cdk5, suggesting that R406W does not inherently increase Ser404 phosphorylation via changes in the actions of these enzymes. Modestly increased phosphorylation of Ser404 was observed by GSK-3β in tau R406W. Collectively, these data suggest a potential role for conformational change to a cis amide bond at Pro405, via Ser404 phosphorylation and/or R406W modification, as a possible mechanism involved in protein misfolding in AD, CTE, and FTDP-17. Alternatively, both Ser404 phosphorylation and the R406W modification lead to increased order, including induced turn formation, in both the trans-proline and cis-proline conformations. 

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