Key Protein Residues Research Articles

Discovery of Novel Lysine Methyltransferase (SMYD3) Inhibitors by Utilizing 3D-QSAR, Molecular Docking and Molecular Dynamics Simulation

Aim: To investigate novel isoxazole amide SMYD3 inhibitors as adjuvant anticancer agents for multiple cancers. Background: SET and MYND Domain-Containing Protein 3 is a hopeful therapeutic target for breast, liver, colon, and prostate cancer. Objective: Novel SMYD3 inhibitors were predicted by the 3D-QSAR models. Methods: In this present work, 3D-QSAR, molecular docking and molecular dynamics (MD) simulations were performed on a series of isoxazole amides-based SMYD3 inhibitors. Results: Molecular docking revealed residues important to protein-compound interactions, indicating that SMYD3 inhibitors have a strong affinity with and bind to key protein residues such as TYR239, MET190, LYS297 and VAL368. The molecular docking results were further validated by molecular dynamics simulations. Conclusion: The above information provided significant guidance for the design of novel SMYD3 inhibitors.

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

#1838 SGLT2 computational structural studies investigating polypharmacy

Abstract Background and Aims Sodium Glucose Cotransporter 2 (SGLT2) is the main active transport protein involved in sodium and glucose reabsorption in the kidney. SGLT2 inhibitors (SGLT2i) are widely recommended for patients with diabetes, heart failure and chronic kidney disease (CKD). Many multimorbid patients will be prescribed these compounds resulting in polypharmacy. We have performed a computational drug repurposing screen to identify other licensed drugs capable of binding at or near the SGLT2i active site aiming to identify compounds that could either compete with SGLT2i or inhibit sodium and glucose transport. Method The library of BNF listed compounds was obtained from NCBI PubChem. D-I-TASSER was used to generate monomeric structural models and MODELLER was used to incorporate MAP-17 and empaglifozin from a reference structure (PDB 7VSI). CHARM-GUI was used to insert the protein into a membrane. The centroid of a 5 nanosecond GROMACS molecular dynamics (MD) equilibration was used for docking studies using PLANTS. A bespoke tool was used to identify compounds interacting with key protein residues. CHARM-GUI and bespoke software were used to prepare membrane-bound systems to run in GROMACS using GPU's in Google Colab. 10 nanosecond simulations were undertaken (300 Kelvin and 1 bar) to discriminate between binding and non-binding events. Results The SGLT2-MAP17 structure in the inward-open conformation obtained showed good agreement with published structures. Existing SGLT2i (empaglifozin, dapaglifozin, canaglifozin, ertuglifozin) all feature in the top 1% of docked compounds in the repurposing screen. 17 compounds were investigated by MD, with all remaining bound to the protein in simulation. Ceftriaxone, tobramycin, clindamycin, fluvastatin, atorvastatin and ticagrelor were among the compounds with potentially significant interactions. It is not clear whether the stable ligand interactions identified here would result in inhibition of sodium and glucose transport, or if the interactions might provide competitive inhibition for SGLT2i compounds currently used. The compounds identified have no hitherto recognised interactions with SGLT2i; nor do they result in adverse effects suggesting inhibition of the protein. This study is limited by considering only the protein-ligand interaction and not wider pharmacokinetic or pharmacodynamic factors. Conclusion The indication of interactions with several compounds likely to be prescribed alongside SGLT2 inhibitors, such as antibiotics, statins and antiplatelet agents, warrants further investigation of the potential for polypharmacological complications.

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

Structure-based prediction of nucleic acid binding residues by merging deep learning- and template-based approaches.

Accurate prediction of nucleic binding residues is essential for the understanding of transcription and translation processes. Integration of feature- and template-based strategies could improve the prediction of these key residues in proteins. Nevertheless, traditional hybrid algorithms have been surpassed by recently developed deep learning-based methods, and the possibility of integrating deep learning- and template-based approaches to improve performance remains to be explored. To address these issues, we developed a novel structure-based integrative algorithm called NABind that can accurately predict DNA- and RNA-binding residues. A deep learning module was built based on the diversified sequence and structural descriptors and edge aggregated graph attention networks, while a template module was constructed by transforming the alignments between the query and its multiple templates into features for supervised learning. Furthermore, the stacking strategy was adopted to integrate the above two modules for improving prediction performance. Finally, a post-processing module dependent on the random walk algorithm was proposed to further correct the integrative predictions. Extensive evaluations indicated that our approach could not only achieve excellent performance on both native and predicted structures but also outperformed existing hybrid algorithms and recent deep learning methods. The NABind server is available at http://liulab.hzau.edu.cn/NABind/.

In silico research on new sulfonamide derivatives as BRD4 inhibitors targeting acute myeloid leukemia using various computational techniques including 3D-QSAR, HQSAR, molecular docking, ADME/Tox, and molecular dynamics

Acute myeloid leukemia, a serious condition affecting stem cells, drives uncontrollable myeloblast proliferation, leading to accumulation. Extensive research seeks rapid, effective chemotherapeutics. A potential option is a BRD4 inhibitor, known for suppressing cell proliferation. Sulfonamide derivatives probed essential structural elements for potent BRD4 inhibitors. To achieve this goal, we employed 3D-QSAR molecular modeling techniques, including CoMFA, CoMSIA, and HQSAR models, along with molecular docking and molecular dynamics simulations. The validation of the 2D/3D QSAR models, both internally and externally, underscores their robustness and reliability. The contour plots derived from CoMFA, CoMSIA, and HQSAR analyses played a pivotal role in shaping the design of effective BRD4 inhibitors. Importantly, our findings highlight the advantageous impact of incorporating bulkier substituents on the pyridinone ring and hydrophobic/electrostatic substituents on the methoxy-substituted phenyl ring, enhancing interactions with the BRD4 target. The interaction mode of the new compounds with the BRD4 receptor (PDB ID: 4BJX) was investigated using molecular docking simulations, revealing favorable binding energies, supported by the formation of hydrogen and hydrophobic bonds with key protein residues. Moreover, these novel inhibitors exhibited good oral bioavailability and demonstrated non-toxic properties based on ADMET analysis. Furthermore, the newly designed compounds along with the most active one from series 58, underwent a molecular dynamics simulation to analyze their behavior. The simulation provided additional evidence to support the molecular docking results, confirming the sustained stability of the analyzed molecules over the trajectory. This outcome could serve as a valuable reference for designing and developing novel and effective BRD4 inhibitors. Communicated by Ramaswamy H. Sarma

Repurposing of World-Approved Drugs for Potential Inhibition against Human Carbonic Anhydrase I: A Computational Study.

Human carbonic anhydrases (hCAs) have enzymatic activities for reversible hydration of CO2 and are acknowledged as promising targets for the treatment of various diseases. Using molecular docking and molecular dynamics simulation approaches, we hit three compounds of methyl 4-chloranyl-2-(phenylsulfonyl)-5-sulfamoyl-benzoate (84Z for short), cyclothiazide, and 2,3,5,6-tetrafluoro-4-piperidin-1-ylbenzenesulfonamide (3UG for short) from the existing hCA I inhibitors and word-approved drugs. As a Zn2+-dependent metallo-enzyme, the influence of Zn2+ ion models on the stability of metal-binding sites during MD simulations was addressed as well. MM-PBSA analysis predicted a strong binding affinity of -18, -16, and -14 kcal/mol, respectively, for these compounds, and identified key protein residues for binding. The sulfonamide moiety bound to the Zn2+ ion appeared as an essential component of hCA I inhibitors. Vina software predicted a relatively large (unreasonable) Zn2+-sulfonamide distance, although the relative binding strength was reproduced with good accuracy. The selected compounds displayed potent inhibition against other hCA isoforms of II, XIII, and XIV. This work is valuable for molecular modeling of hCAs and further design of potent inhibitors.

Conserved Conformational Dynamics Reveal a Key Dynamic Residue in the Gatekeeper Loop of Human Cyclophilins.

Cyclophilins are ubiquitous human enzymes that catalyze peptidyl-prolyl cis-trans isomerization in protein substrates. Of the 17 unique isoforms, five closely related isoforms (CypA-E) are found in various environments and participate in diverse cellular processes, yet all have similar structures and the same core catalytic function. The question is what key residues are behind the conserved function of these enzymes. Here, conformational dynamics are compared across these isoforms to detect conserved dynamics essential for the catalytic activity of cyclophilins. A set of key dynamic residues, defined by the most dynamically conserved positions, are identified in the gatekeeper 2 region. The highly conserved glycine (Gly80) in this region is predicted to underlie the local flexibility, which is further tested by molecular dynamics simulations performed on mutants (G80A) of CypE and CypA. The mutation leads to decreased flexibility of CypE and CypA during substrate binding but increased flexibility during catalysis. Dynamical changes occur in the mutated region and a distal loop downstream of the mutation site in sequence. Examinations of the mutational effect on catalysis show that both mutated CypE and CypA exhibit shifted binding free energies of the substrate under distinct isomer conformations. The results suggest a loss of function in the mutated CypE and CypA. These catalytic changes by the mutation are likely independent of the substrate sequence, at least in CypA. Our work presents a method to identify function-related key residues in proteins.

Hydroxyalkynyl uracil derivatives as NNRTIs against HIV-1: in silico predictions, synthesis, docking and molecular dynamics simulation studies

To improve rationally the efficacy of the non-nucleoside human immunodeficiency virus (HIV-1) inhibitors, it is important to have a precise and detailed understanding of the HIV-1 reverse transcriptase (RT) and inhibitor interactions. For the 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio) thymine (HEPT) type of nucleoside reverse transcriptase inhibitors (NNRTIs), the H-bond between the N-3H of the inhibitor and the backbone carbonyl group of K101 represents the major hydrophilic interaction. This H-bond contributes to the NNRTI binding affinity. The descriptor analyses of different uracil derivatives proved their good cell internalization. The bioactivity score reflected higher drug likeness score and the ligands showed interesting docking results. All molecules were deeply buried and stabilized into the allosteric site of HIV-1 RT. For majority of molecules, residues Lys101, Lys103, Tyr181 and Tyr188 were identified as key protein residues responsible for generation of H-bond and major interactions were similar to all known NNRTIs while very few molecules interacted with residues Phe227 and Tyr318. The TOPKAT protocol available in Discovery Studio 3.0 was used to predict the pharmacokinetics of the designed uracil derivatives in the human body. The molecular dynamics (MD) and post-MD analyses results reflected that the complex HIVRT:5 appeared to be more stable than the complex HIVRT:HEPT, where HEPT was used as reference. Different uracil derivatives have been synthesized by using uracil as starting material and commercially available propargyl bromide. The N-1 derivative of uracil was further reacted with sodamide and different aldehydes/ketones bearing alkyl and phenyl ring to obtain hydroxyalkynyl uracil derivatives as NNRTIs. Communicated by Ramaswamy H. Sarma

Covariation analysis of the master bacterial DNA replication initiator DnaA

Bacterial DNA replication initiation is governed by DnaA: an oligomeric protein belonging to the ATPases associated with diverse cellular activities (AAA+) superfamily. The master replication initiator is present in all known bacterial pathogens; thus, it is an attractive target for drug discovery. Here, an in-silico evolutionary covariation analysis of DnaA amino acid residues was conducted using EVcouplings, a Python framework, to investigate connections between key protein residues. Several co-evolving amino acid pairs were identified and characterized using PyMol to visualize the residues on DnaA crystal structures. I hypothesize that some co-evolving pairs, particularly those spatially connected, are likely important for structural integrity of the protein. Interestingly, one co-evolving pair of residues (A. aeolicus Val121 and Ser229) was found to be separated by ~27 angstroms, suggesting a more complex relationship connects these two sites. Thus, analysing co-evolution of DnaA residues supports known structural information and may predict either allosteric connections within DnaA or contacts between DnaA and other binding partners.

Mechanistic insights of key host proteins and potential repurposed inhibitors regulating SARS-CoV-2 pathway.

The emergence of pandemic situations originated from severe acute respiratory syndrome (SARS)‐CoV‐2 and its new variants created worldwide medical emergencies. Due to the non‐availability of efficient drugs and vaccines at these emergency hours, repurposing existing drugs can effectively treat patients critically infected by SARS‐CoV‐2. Finding a suitable repurposing drug with inhibitory efficacy to a host‐protein is challenging. A detailed mechanistic understanding of the kinetics, (dis)association pathways, key protein residues facilitating the entry–exit of the drugs with targets are fundamental in selecting these repurposed drugs. Keeping this target as the goal of the paper, the potential repurposing drugs, Nafamostat, Camostat, Silmitasertib, Valproic acid, and Zotatifin with host‐proteins HDAC2, CSK22, eIF4E2 are studied to elucidate energetics, kinetics, and dissociation pathways. From an ensemble of independent simulations, we observed the presence of single or multiple dissociation pathways with varying host‐proteins‐drug systems and quantitatively estimated the probability of unbinding through these specific pathways. We also explored the crucial gateway residues facilitating these dissociation mechanisms. Interestingly, the residues we obtained for HDAC2 and CSK22 are also involved in the catalytic activity. Our results demonstrate how these potential drugs interact with the host machinery and the specific target residues, showing involvement in the mechanism. Most of these drugs are in the preclinical phase, and some are already being used to treat severe COVID‐19 patients. Hence, the mechanistic insight presented in this study is envisaged to support further findings of clinical studies and eventually develop efficient inhibitors to treat SARS‐CoV‐2.

Role of key protein residues involved in DinB DNA damage specificity: an in silico study

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb, and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little finger” or polymerase‐associated domain (PAD) and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. Specifically, we analyzed a lesion that the protein can bypass (N2‐furfuryl‐dG) and one that instead the protein does not bypass well (O6‐Methylated dG). Our MD simulations revealed that there are some key residues in the fingers domain that are important switches for the conformational change. For example, R35 assumes a characteristic position when the protein is in a catalytically competent state, while it blocks the active site when the protein is not able to bypass the lesion. This result is in agreement with previous studies which showed that mutations of the R35 residue alter the protein specificity. Other areas of DinB which seem to undergo large movement reside in the PAD domain. This domain, which makes contact with the DNA major groove, has been implicated in DNA damage specificity, so it makes sense that residues on this domain assume different conformations, depending on the type of lesions present on the templating strand. A discussion of possible roles for some of the residues on the PAD will be presented.

In silico analysis of kiss2, expression studies and protein–protein interaction with gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHβ) in Heteropneustes fossilis

Kisspeptins, encoded by the kiss genes, are neuropeptides that regulate the onset of puberty, maturation of gonads, and fertility in higher vertebrates including fishes. The gene ontology suggests that kisspeptin plays an important role not only in reproduction but also in cell signaling, immune response and metabolic processes, and to decipher protein–protein interactions, computational approach has been favored. The present investigation focuses on the detailed structural analysis and molecular docking of kiss2 gene using in silico tools. A putative kiss2 protein of 113 amino acids was encoded by an open reading frame of 342 bp kiss2 gene. The protein is of 13.12 kDa with isoelectric point of 9.45. The secondary structure of the protein indicates more than 50% random coils, followed by 34% of alpha helix and 13% extended strand. The protein was found to be extracellular and secretory in nature. Since, protein–protein interactions play a very crucial role in every cellular process and due to unavailability of crystal structure of our protein of interest in fishes computational approach has been employed. The 3D PDB modeling and the molecular docking of kiss2, Gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHβ) proteins in fishes have been demonstrated applying protein-docking approach. Molecular interactions of kiss2 protein were the highest with kisspeptin receptor 2 and lowest for the neuropeptide FF-amide peptide precursor protein. Expression of kiss2 transcripts, mainly in the brain and ovary of H. fossilis, supports its hypothalamic–pituitary–gonadal axis signaling and reproductive function. Further, changes in expression patterns of kiss2 mRNA during different developmental stages, indicate its potential role in embryonic development also. The present study conclusively reveals interaction of kiss2 with other neuropeptides. Prediction of binding structures and identification of key residues in protein–protein interaction illustrate direct interaction among these proteins, playing a cardinal role in neuroendocrine regulation of reproduction in catfish. Communicated by Ramaswamy H. Sarma

Target-Specific Prediction of Ligand Affinity with Structure-Based Interaction Fingerprints.

Discovery and optimization of small molecule inhibitors as therapeutic drugs have immensely benefited from rational structure-based drug design. With recent advances in high-resolution structure determination, computational power, and machine learning methodology, it is becoming more tractable to elucidate the structural basis of drug potency. However, the applicability of machine learning models to drug design is limited by the interpretability of the resulting models in terms of feature importance. Here, we take advantage of the large number of available inhibitor-bound HIV-1 protease structures and associated potencies to evaluate inhibitor diversity and machine learning models to predict ligand affinity. First, using a hierarchical clustering approach, we grouped HIV-1 protease inhibitors and identified distinct core structures. Explicit features including protein-ligand interactions were extracted from high-resolution cocrystal structures as 3D-based fingerprints. We found that a gradient boosting machine learning model with this explicit feature attribution can predict binding affinity with high accuracy. Finally, Shapley values were derived to explain local feature importance. We found specific van der Waals (vdW) interactions of key protein residues are pivotal for the predicted potency. Protein-specific and interpretable prediction models can guide the optimization of many small molecule drugs for improved potency.

Identification of Key Residues in Proteins Through Centrality Analysis and Flexibility Prediction with RINspector

Protein structures inherently contain information that can be used to decipher their functions, but the exploitation of this knowledge is not trivial. We recently developed an app for the Cytoscape network visualization and analysis program, called RINspector, the goal of which is to integrate two different approaches that identify key residues in a protein structure or complex. The first approach consists of calculating centralities on a residue interaction network (RIN) generated from the three-dimensional structure; the second consists of predicting backbone flexibility and needs only the primary sequence. The identified residues are highly correlated with functional relevance and constitute a good set of targets for mutagenesis experiments. Here we present a protocol that details in a step-by-step fashion how to create a RIN from a structure and then calculate centralities and predict flexibilities. We also discuss how to understand and use the results of the analyses. © 2018 by John Wiley & Sons, Inc.

Identification and Analysis of Key Residues in Protein-RNA Complexes.

Protein-RNA complexes play important roles in various biological processes. The functions of protein-RNA complexes are dictated by their interactions, binding, stability, and affinity. In this work, we have identified the key residues (KRs), which are involved in both stability and binding. We found that 42 percent of considered proteins share common binding and stabilizing residues, whereas these residues are distinct in 58 percent of the proteins. Overall, 5 percent of stabilizing and 3 percent of binding residues serve as key residues. These residues are enriched with the combination of polar, charged, aliphatic, and aromatic residues. Analysis on subclasses of protein-RNA complexes based on protein structural class, function and RNA type showed that regulatory proteins, and complexes with single stranded RNA and rRNA have appreciable number of key residues. Specifically, Arg, Tyr, and Thr are preferred in most of the subclasses of protein-RNA complexes. In addition, residues with similar chemical behavior have different preferences to be KRs, such that Arg, Tyr, Val, and Thr are preferred over Lys, Trp, Ile, and Ser, respectively. Atomic level contacts revealed that charged and polar-nonpolar contacts are dominant in enzymes, polar in structural, and nonpolar in regulatory proteins. On the other hand, polar-nonpolar contacts are enriched in all these classes of protein-RNA complexes. Further, the influence of sequence and structural features such as conservation score, surrounding hydrophobicity, solvent accessibility, secondary structure, and long-range order in key residues are also discussed. We envisage that the present study provides insights to understand the structural and functional aspects of protein-RNA complexes.

Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Structural Insights into Histone Crotonyl-Lysine Recognition by the AF9 YEATS Domain

Histone lysine acylations play an important role in theregulation of gene transcription in chromatin. Unlike histone acetyl-lysine, molecular recognition of a recently identified crotonyl-lysine mark is much less understood. Here, we report that the YEATS domain of AF9 preferentially binds crotonyl-lysine over acetyl-lysine in histone H3. Nuclear magnetic resonance structural analysis reveals that crotonyl-lysine of histone H3 lysine 18 is engulfed deep in an aromatic cage of the YEATS domain where the carbonyl oxygen of crotonyl-lysine forms a hydrogen bond with the backbone amide of protein residue Tyr78. The crotonyl-lysine, through its unique electron-rich double-bond side chain, engages π-π aromatic stacking and extended hydrophobic/aromatic interactions with the YEATS domain compared with acetyl-lysine. Our mutational analysis confirmed key protein residues Phe59 and Tyr78 for crotonyl-lysine recognition. Importantly, our findings present a new structural mechanism of protein-protein interactions mediated by histone lysine crotonylation, and show how the cells interpret acyl-lysine marks in different biological contexts.

Sensitivity in Binding Free Energies Due to Protein Reorganization

Tremendous recent improvements in computer hardware, coupled with advances in sampling techniques and force fields, are now allowing protein-ligand binding free energy calculations to be routinely used to aid pharmaceutical drug discovery projects. However, despite these recent innovations, there are still needs for further improvement in sampling algorithms to more adequately sample protein motion relevant to protein-ligand binding. Here, we report our work identifying and studying such clear and remaining needs in the apolar cavity of T4 lysozyme L99A. In this study, we model recent experimental results that show the progressive opening of the binding pocket in response to a series of homologous ligands.1 Even while using enhanced sampling techniques, we demonstrate that the predicted relative binding free energies (RBFE) are sensitive to the initial protein conformational state. Particularly, we highlight the importance of sufficient sampling of protein conformational changes and demonstrate how inclusion of three key protein residues in the "hot" region of the FEP/REST simulation improves the sampling and resolves this sensitivity, given enough simulation time.

Deazaflavin Inhibitors of Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Specific for the Human Enzyme and Active against Cellular TDP2.

Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively inhibit the human TDP2 enzyme in a competitive manner both with recombinant and native TDP2. We show that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to the drugs and that key protein residues are responsible for drug resistance. Among them, human residues L313 and T296 confer high resistance when mutated to their mouse counterparts. Moreover, deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in human prostate cancer DU145 cells and TDP2-dependent synergy in TK6 human lymphoblast and avian DT40 cells. Deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors.