ABSTRACT
UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.
Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin , Ubiquitin-Conjugating Enzymes/metabolism , Binding SitesABSTRACT
UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.
Subject(s)
Cysteine , Ubiquitin , Ubiquitin/metabolism , Cysteine/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , High-Throughput Screening AssaysABSTRACT
Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.
Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin , Humans , Nuclear Magnetic Resonance, Biomolecular , Ubiquitination , Ubiquitin/metabolismABSTRACT
Dengue virus is an important human pathogen affecting people especially in tropical and subtropical regions. Its genome encodes seven non-structural proteins that are important for viral assembly and replication. Dengue NS2B is a membrane protein containing four transmembrane helices and involved in protein-protein interactions. Its transmembrane helices are critical for location of NS2B on the cell membrane while one cytoplasmic region composed of approximately 40 amino acids serves as a cofactor of viral NS3 protease by forming a tight complex with the N-terminal region of NS3. Here, we report the backbone resonance assignments for a dengue NS2B construct referred to as mini-NS2B containing only the transmembrane regions without NS3 cofactor region in detergent micelles. Mini-NS2B exhibits well-dispersed cross-peaks in the 1H-15N-HSQC spectrum and contains four helices in solution. The available mini-NS2B and its assignment will be useful for determining the structure of NS2B and identifying small molecules binding to the transmembrane regions.
Subject(s)
Dengue , Peptide Hydrolases , Humans , Micelles , Detergents/chemistry , Nuclear Magnetic Resonance, Biomolecular , Viral Nonstructural Proteins/chemistryABSTRACT
Cellular inhibitor of apoptosis protein-1 (cIAP-1) is member of inhibitor of apoptosis proteins (IAPs) which can affect apoptosis through interactions with caspases. cIAP-1 is a multi-domain protein and able to regulate apoptosis through interactions with proteins such as caspases and possesses E3 ligase activity. Human cIAP-1 contains three baculovirus IAP repeat (BIR) domains which are critical for protein-protein interactions. Here, we report NMR resonance assignments of the first BIR domain of human cIAP. Its secondary structures in solution were determined based on the assigned resonances. The dynamics of this domain was obtained, and our hydrogen-deuterium exchange experiment reveals that the first helix in BIR1 is exposed to the solvent. The availability of assignments of backbone and side chain resonances will be useful for probing protein-protein interactions.
Subject(s)
Baculoviridae , Inhibitor of Apoptosis Proteins , Apoptosis/physiology , Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Baculoviridae/metabolism , Caspases/metabolism , Cell Line , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein BindingABSTRACT
Bacterial tRNA (guanine37-N1)-methyltransferase (TrmD) plays important roles in translation, making it an important target for the development of new antibacterial compounds. TrmD comprises two domains with the N-terminal domain binding to the S-adenosyl-L-methionine (SAM) cofactor and the C-terminal domain critical for tRNA binding. Bacterial TrmD is functional as a dimer. Here we report the backbone NMR resonance assignments for the full length TrmD protein of Pseudomonas aeruginosa. Most resonances were assigned and the secondary structure for each amino acid was determined according to the assigned backbone resonances. The availability of the assignment will be valuable for exploring molecular interactions of TrmD with ligands, inhibitors and tRNA.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Pseudomonas aeruginosa/enzymology , tRNA Methyltransferases/chemistry , Models, Molecular , Protein Domains , tRNA Methyltransferases/metabolismABSTRACT
Syndecans are single-span membrane proteins playing important roles in cell-cell and cell-matrix interactions. The transmembrane domain of syndecans is critical for signal transduction across the cell membrane. Here, the structure of the transmembrane domain of syndecan-2 in detergent micelles was investigated using solution NMR spectroscopy. Backbone resonance assignment was obtained, and NMR studies show that the transmembrane domain forms a helix in detergent micelles, which is also supported by the hydrogen and deuterium exchange experiment. A study of the dynamics revealed the rigid structure of the transmembrane domain formed in solution, and paramagnetic relaxation enhancement defined the topology of the transmembrane domain in detergent micelles. This structural analysis may facilitate a better understanding of the role of the syndecan-2 transmembrane domain in signal transduction.
Subject(s)
Detergents/chemistry , Micelles , Syndecan-2/chemistry , Cell Membrane/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Syndecan-2/metabolismABSTRACT
Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N1G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , RNA, Transfer/metabolism , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Discovery , Enzyme Inhibitors/chemistry , Kinetics , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Pseudomonas aeruginosa/genetics , Substrate SpecificityABSTRACT
Bacterial tRNA (guanine37-N1)-methyltransferase (TrmD) is an important antibacterial target due to its essential role in translation. TrmD has two domains connected with a flexible linker. The N-terminal domain (NTD) of TrmD contains the S-adenosyl-L-methionine (SAM) cofactor binding site and the C-terminal domain is critical for tRNA binding. Here we report the backbone NMR resonance assignments for NTD of Pseudomonas aeruginosa TrmD. Its secondary structure was determined based on the assigned resonances. Relaxation analysis revealed that NTD existed as dimers in solution. NTD also exhibited thermal stability in solution. Its interactions with SAM and other compounds suggest it can be used for evaluating SAM competitive inhibitors by NMR.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Pseudomonas aeruginosa/enzymology , tRNA Methyltransferases/chemistry , Ligands , Protein DomainsABSTRACT
NSD3 is a histone H3 methyltransferase that plays an important role in chromatin biology. A construct containing the methyltransferase domain encompassing residues Q1049-K1299 of human NSD3 was obtained and biochemical activity was demonstrated using histone as a substrate. Here we report the backbone HN, N, Cα, C', and side chain Cß assignments of the construct in complex with S-adenosyl-L-methionine (SAM). Based on these assignments, secondary structures of NSD3/SAM complex in solution were determined.
Subject(s)
Coenzymes/metabolism , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , PR-SET Domains , Amino Acid Sequence , HumansABSTRACT
Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.
Subject(s)
DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/chemistry , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Topoisomerase Inhibitors/chemistry , Humans , Indoles/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Domains , Protein Structure, SecondaryABSTRACT
The human ether-à-go-go related gene (hERG) channel is crucial for the cardiac action potential by contributing to the fast delayed-rectifier potassium current. Mutations in the hERG channel result in type 2 long QT syndrome (LQT2). The hERG channel contains a cyclic nucleotide-binding homology domain (CNBHD) and this domain is required for the channel gating though molecular interactions with the eag domain. Here we present solution structure of the CNBHD of the hERG channel. The structural study reveals that the CNBHD adopts a similar fold to other KCNH channels. It is self-liganded and it contains a short ß-strand that blocks the nucleotide-binding pocket in the ß-roll. Folding of LQT2-related mutations in this domain was shown to be affected by point mutation. Mutations in this domain can cause protein aggregation in E. coli cells or induce conformational changes. One mutant-R752W showed obvious chemical shift perturbation compared with the wild-type, but it still binds to the eag domain. The helix region from the N-terminal cap domain of the hERG channel showed unspecific interactions with the CNBHD.
Subject(s)
ERG1 Potassium Channel/chemistry , Long QT Syndrome/genetics , Binding Sites , ERG1 Potassium Channel/genetics , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutation, Missense , Point Mutation , Protein Conformation, alpha-Helical , Protein Folding , Protein Interaction Domains and Motifs , SolutionsABSTRACT
Bacterial DNA topoisomerases are important drug targets due to their importance in DNA replication and low homology to human topoisomerases. The N-terminal 24 kDa region of E. coli topoisomerase IV E subunit (eParE) contains the ATP binding pocket. Structure-based drug discovery has been proven to be an efficient way to develop potent ATP competitive inhibitors against ParEs. NMR spectroscopy is a powerful tool to understand protein and inhibitor interactions in solution. In this study, we report the backbone assignment for the N-terminal domain of E. coli ParE. The secondary structural information and the assignment will aid in structure-based antibacterial agents development targeting eParE.
Subject(s)
DNA Topoisomerase IV/chemistry , Escherichia coli/enzymology , Nuclear Magnetic Resonance, Biomolecular , Protein Subunits/chemistry , Anti-Bacterial Agents/pharmacology , DNA Topoisomerase IV/metabolism , Drug Discovery , Escherichia coli/drug effects , Protein Structure, Secondary , Protein Subunits/metabolismABSTRACT
Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between ß2 and α3, and the ß2 and ß6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.
Subject(s)
DNA Topoisomerase IV/chemistry , Amino Acid Sequence , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Pseudomonas aeruginosa , Solutions , Streptococcus pneumoniae , Surface Plasmon Resonance , TemperatureABSTRACT
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.
Subject(s)
Adenosine Triphosphate/metabolism , DNA Topoisomerase IV/metabolism , DNA Topoisomerase IV/pharmacology , Escherichia coli/enzymology , Adenosine Triphosphate/antagonists & inhibitors , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Binding, Competitive , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/chemistry , Drug Design , Molecular Sequence Data , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.
Subject(s)
Catalytic Domain , DNA Gyrase/chemistry , DNA Gyrase/metabolism , Pseudomonas aeruginosa/enzymology , Topoisomerase II Inhibitors/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Urea/chemistry , Urea/metabolism , Urea/pharmacologyABSTRACT
Bacterial resistance to antibiotics remains a serious threat to global health. The gyraseâ B enzyme is a well-validated target for developing antibacterial drugs. Despite being an attractive target for antibiotic development, there are currently no gyraseâ B inhibitory drugs on the market. A fragment screen using 1,800â compounds identified 14â fragments that bind to Escherichia coli (E.â coli) gyraseâ B. The detailed characterization of binding is described for all 14â fragments. With the aid of X-ray crystallography, modifications on a low-affinity fragment (KD =253â µM, IC50 =634â µM) has led to the development of a new class of potent phenyl aminopyrazole inhibitors against E.â coli gyraseâ B (IC50 =160â nM). The study presented here combines the use of a set of biophysical techniques including differential scanning fluorimetry, nuclear magnetic resonance, isothermal titration calorimetry, and X-ray crystallography to methodically identify, quantify, and optimize fragments into new chemical leads.
ABSTRACT
The KCNH family of ion channels plays important roles in heart and nerve cells. The C-terminal region of the KCNH channel contains a cyclic-nucleotide binding homology domain (CNBHD) which is important for channel gating through interaction with the eag domain. To study the solution structure of CNBHD of the KCNH channel of zebrafish, we over-expressed and purified this domain from E. coli. We report the resonance assignments of the CNBHD. The assignments will allow us to perform structural and dynamic studies for this domain, which will shed light on its role in channel gating.
Subject(s)
Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nucleotides, Cyclic/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism , Animals , Protein Structure, Tertiary , ZebrafishABSTRACT
The hERG (human ether-a-go-go related gene) potassium channel is a voltage-gated potassium channel containing an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2-S3 linker, S3, S4 and the S4-S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4-S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3-S4 and S4-S4-S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating.
Subject(s)
Ether-A-Go-Go Potassium Channels/chemistry , Amino Acid Sequence , Chromatography, Gel , ERG1 Potassium Channel , Micelles , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Spectrometry, FluorescenceABSTRACT
The gating of the hERG channel is regulated by its eag domain through molecular interaction with either the cyclic nucleotide-binding homology domain (CNBHD) or the linker between transmembrane segments 4 and 5. Our NMR study on the purified CNBHD demonstrated that it contains nine ß-strands and does not bind cAMP. We show that the eag domain binds to the CBND through an interface containing several disease-associated mutations. The N-terminal cap domain and R56 in the eag domain are important for the interaction with the CNBHD. Residues from the CNBHD that were affected by the interaction with the eag domain were also identified. A R56Q mutation does not cause major structural changes in the eag domain and showed reduced interaction with the CNBHD.