The binding mode of orphan glycyl-tRNA synthetase with tRNA supports the synthetase classification and reveals large domain movements.

As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.

Structure-Guided Design of Halofuginone Derivatives as ATP-Aided Inhibitors Against Bacterial Prolyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are promising antimicrobial targets due to their essential roles in protein translation, and expanding their inhibitory mechanisms will provide new opportunities for drug discovery. We report here that halofuginone (HF), an herb-derived medicine, moderately inhibits prolyl-tRNA synthetases (ProRSs) from various pathogenic bacteria. A cocrystal structure of Staphylococcus aureus ProRS (SaProRS) with HF and an ATP analog was determined, which guided the design of new HF analogs. Compound 3 potently inhibited SaProRS at IC50 = 0.18 µM and Kd = 30.3 nM and showed antibacterial activities with an MIC of 1-4 µg/mL in vitro. The bacterial drug resistance to 3 only developed at a rate similar to or slower than those of clinically used antibiotics in vitro. Our study indicates that the scaffold and ATP-aided inhibitory mechanism of HF could apply to bacterial ProRS and also provides a chemical validation for using bacterial ProRS as an antibacterial target.

X-shaped structure of bacterial heterotetrameric tRNA synthetase suggests cryptic prokaryote functions and a rationale for synthetase classifications.

AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding ß-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and ß-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain ß-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family.

Identification of new building blocks by fragment screening for discovering GyrB inhibitors.

DNA gyrase is an essential DNA topoisomerase that exists only in bacteria. Since novobiocin was withdrawn from the market, new scaffolds and new mechanistic GyrB inhibitors are urgently needed. In this study, we employed fragment screening and X-ray crystallography to identify new building blocks, as well as their binding mechanisms, to support the discovery of new GyrB inhibitors. In total, 84 of the 618 chemical fragments were shown to either thermally stabilize the ATPase domain of Escherichia coli GyrB or inhibit the ATPase activity of E. coli gyrase. Among them, the IC50 values of fragments 10 and 23 were determined to be 605.3 µM and 446.2 µM, respectively. Cocrystal structures of the GyrB ATPase domain with twelve fragment hits were successfully determined at a high resolution. All twelve fragments were deeply inserted in the pocket and formed H-bonds with Asp73 and Thr165, and six fragments formed an additional H-bond with the backbone oxygen of Val71. Fragment screening further highlighted the capability of Asp73, Thr165 and Val71 to bind chemicals and provided diverse building blocks for the design of GyrB inhibitors.

Structural insights into the ligand recognition and catalysis of the key aminobutanoyltransferase CntL in staphylopine biosynthesis.

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process.

Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase.

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

Structure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) are an attractive class of antibacterial drug targets due to their essential roles in protein translation. While most traditional aaRS inhibitors target the binding pockets of substrate amino acids and/or ATP, we recently developed a class of novel tRNA-amino acid dual-site inhibitors including inhibitor 3 ((2S,3R)-2-amino-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide) against threonyl-tRNA synthetase (ThrRS). Here, the binding modes and structure-activity relationships (SARs) of these inhibitors were analyzed by the crystal structures of Salmonella enterica ThrRS (SeThrRS) in complex with three of them. Based on the cocrystal structures, twelve quinazolinone-threonine hybrids were designed and synthesized, and their affinities, enzymatic inhibitory activities, and cellular potencies were evaluated. The best derivative 8g achieved a Kd value of 0.40 µM, an IC50 value of 0.50 µM against SeThrRS and MIC values of 16-32 µg/mL against the tested bacterial strains. The cocrystal structure of the SeThrRS-8g complex revealed that 8g induced a bended conformation for Met332 by forming hydrophobic interactions, which better mimicked the binding of tRNAThr to ThrRS. Moreover, the inhibitory potency of 8g was less impaired than a reported ATP competitive inhibitor at high concentrations of ATP, supporting our hypothesis that tRNA site inhibitors are likely superior to ATP site inhibitors in vivo, where ATP typically reaches millimolar concentrations.

Structural and Biochemical Characterization of SbnC as a Representative Type B Siderophore Synthetase.

Staphyloferrin B is a key siderophore secreted by Staphylococcus aureus to acquire ferric ions from a host during infection, and its biosynthetic pathway has been validated to develop efficient antibacterial agents. Herein, we report the crystal structure of AMP-bound SbnC from S. aureus (SaSbnC) as the first representative structure of type B synthetases in the biosynthesis of α-hydroxycarboxylate siderophores. While type B synthetases specifically use α-ketoglutarate (α-KG) as their carboxylic acid substrate, SaSbnC showed unique structural features in the substrate pocket compared with the type A and C synthetases. Screening of α-KG analogues suggested that the hydrogen-bonding interaction between the α-carbonyl group of α-KG and residue Lys552 is a key determinant for the substrate selectivity of type B synthetases. Interestingly, citrate, the product of the tricarboxylic acid cycle and the substrate of type A synthetases, was found to inhibit the activity of SaSbnC with an IC50 value of 83 µM by mimicking α-KG binding, suggesting a potential regulatory role of the tricarboxylic acid cycle, whose activity is under the control of the intracellular iron concentration, to SaSbnC and other type B synthetases. These results provide critical new information to understand the structure, function, and regulation of type B synthetases in the siderophore-based iron acquisition system employed by a large number of pathogenic microbes.

Discovery of novel tRNA-amino acid dual-site inhibitors against threonyl-tRNA synthetase by fragment-based target hopping.

Threonyl-tRNA synthetase (ThrRS) is a key member of the aminoacyl-tRNA synthetase (aaRS) family that plays essential roles in protein biosynthesis, and ThrRS inhibitors have potential in the therapy of multiple diseases, such as microbial infections and cancers. Based on a unique tRNA-amino acid dual-site inhibitory mechanism identified recently with the herb-derived prolyl-tRNA synthetase (ProRS) inhibitor halofuginone (HF), a series of compounds have been designed and synthesized by employing a fragment-based target hopping approach to simultaneously target the tRNAThr and l-threonine binding pockets of ThrRS. Among them, compound 30d showed an IC50 value of 1.4 µM against Salmonella enterica ThrRS (SeThrRS) and MIC values of 16-32 µg/mL against the tested bacterial strains. The cocrystal structure of SeThrRS in complex with 30d was determined at high resolution, revealing that 30d simultaneously occupies both binding pockets for the nucleotide A76 of tRNAThr and l-threonine in an ATP-independent manner, a novel mechanism compared to all other reported ThrRS inhibitors. Our study provides a new class of ThrRS inhibitors, and more importantly, it presents the first experimental evidence that the tRNA-amino acid dual-site inhibitory mechanism could apply to other aaRSs beyond ProRS, thus providing great opportunities for designing new mechanistic inhibitors for aaRS-based therapeutics.

Structural Insights into Substrate Recognition and Activity Regulation of the Key Decarboxylase SbnH in Staphyloferrin B Biosynthesis.

Staphyloferrin B is a hydroxycarboxylate siderophore that is crucial for the invasion and virulence of Staphylococcus aureus in mammalian hosts where free iron ions are scarce. The assembly of staphyloferrin B involves four enzymatic steps, in which SbnH, a pyridoxal 5'-phosphate (PLP)-dependent decarboxylase, catalyzes the second step. Here, we report the X-ray crystal structures of S. aureus SbnH (SaSbnH) in complex with PLP, citrate, and the decarboxylation product citryl-diaminoethane (citryl-Dae). The overall structure of SaSbnH resembles those of the previously reported PLP-dependent amino acid decarboxylases, but the active site of SaSbnH showed unique structural features. Structural and mutagenesis analysis revealed that the citryl moiety of the substrate citryl-l-2,3-diaminopropionic acid (citryl-l-Dap) inserts into a narrow groove at the dimer interface of SaSbnH and forms hydrogen bonding interactions with both subunits. In the active site, a conserved lysine residue forms an aldimine linkage with the cofactor PLP, and a phenylalanine residue is essential for accommodating the l-configuration Dap of the substrate. Interestingly, the freestanding citrate molecule was found to bind to SaSbnH in a conformation inverse to that of the citryl group of citryl-Dae and efficiently inhibit SaSbnH. As an intermediate in the tricarboxylic acid (TCA) cycle, citrate is highly abundant in bacterial cells until iron depletion; thus, its inhibition of SaSbnH may serve as an iron-dependent regulatory mechanism in staphyloferrin B biosynthesis.

Identify liver X receptor ß modulator building blocks by developing a fluorescence polarization-based competition assay.

The liver X receptors (LXRs) of the nuclear receptor family are promising therapeutic targets of multiple diseases like lipid disorders, chronic inflammation, as well as different human cancers. To date, no LXR agonists or antagonists can be used in clinics, emphasizing the importance for discovering new LXR modulators. Fragment-based lead discovery (FBLD) is powerful for designing new scaffolds and new mechanistic drugs, but fragment screening has not been applied to LXRs yet, which might be due to the lack of a specific fragment screening method against the dynamic and hydrophobic ligand binding domain (LBD) of LXRs. Herein, a series of fluorescent tracers were designed, synthesized and tested. The tracer based on hyodeoxycholic acid exhibited a good capability for competitively detecting the ligand binding of LXRß using a fluorescence polarization approach. Then, 1074 fragments were screened against the LBD of LXRß (LXRß-LBD), resulting in 27 binding hits. These fragment hits were further tested using the co-activator recruitment assay and reporter gene assay, and efforts in X-ray crystallography fortunately solved a co-crystal structure of LXRß-LBD with the fragment F3 (tert-butyl-7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate). The fluorescence-based fragment screening tool and the newly identified LXRß binding fragments provide the basis for developing novel LXRß modulators.

Crystal structure of CntK, the cofactor-independent histidine racemase in staphylopine-mediated metal acquisition of Staphylococcus aureus.

Staphylopine is a newly identified broad-spectrum metallophore for metal acquisition, and it plays important roles in the survival and virulence of Staphylococcus aureus and other pathogens in the metal-scarce environment in hosts. CntK catalyzes the first step of staphylopine synthesis by converting L-histidine to D-histidine to provide an essential building block of staphylopine. Herein, the crystal structures of S. aureus CntK (SaCntK) and its C72S variant are determined at 1.82 and 1.58 Šresolution, respectively. SaCntK forms a homodimer and each subunit contains a two-domain α/ß structure. Its overall structure resembles diaminopimelate epimerase, although their sequence identities are lower than 22%. SaCntK is specific for histidine, whereas other proteinogenic amino acids, with the exception of arginine, do not show any binding with SaCntK. Structural modeling suggested that residues Asn16, Glu46, Gln47 and Glu208 are responsible for specific substrate binding, and their substitutions significantly reduced the binding of histidine to SaCntK. Structural modeling suggested SaCntK uses a two-base catalytic mechanism, which has been observed in many cofactor-independent racemases. Our study provides critical insights into the structure and functions of CntK in staphylopine synthesis, which makes it helpful for developing potential antibiotics targeting the staphylopine-mediated metal acquisition process in bacteria.

Discovery of indoleamine 2,3-dioxygenase inhibitors using machine learning based virtual screening.

Indoleamine 2,3-dioxygenase (IDO), an immune checkpoint, is a promising target for cancer immunotherapy. However, current IDO inhibitors are not approved for clinical use yet; therefore, new IDO inhibitors are still demanded. To identify new IDO inhibitors, we have built naive Bayesian (NB) and recursive partitioning (RP) models from a library of known IDO inhibitors derived from recent publications. Thirteen molecular fingerprints were used as descriptors for the models to predict IDO inhibitors. An in-house compound library was virtually screened using the best machine learning model, which resulted in 50 hits for further enzyme-based IDO inhibitory assays. Consequently, we identified three new IDO inhibitors with IC50 values of 1.30, 4.10, and 4.68 µM. These active compounds also showed IDO inhibitory activities in cell-based assays. The compounds belong to the tanshinone family, a typical scaffold family derived from Danshen (a Chinese herb), the dried root of Salvia miltiorrhiza, which has been widely used in China, Japan, the United States, and other European countries for the treatment of cardiovascular and cerebrovascular diseases. Thus, we discovered a new use for Danshen using machine learning methods. Surface plasmon resonance (SPR) experiments proved that the inhibitors interacted with the IDO target. Molecular dynamic simulations demonstrated the binding modes of the IDO inhibitors.

Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity.

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.

Pseudellones A-C, Three Alkaloids from the Marine-Derived Fungus Pseudallescheria ellipsoidea F42-3.

Pseudellones A and B (1 and 2), a pair of irregularly bridged epimonothiodiketopiperazine diastereomers constructed from unusual 3-indolylglycine and alanine residues, and an alkaloid pseudellone C (3) possessing a unique skeleton were isolated from the marine-derived fungus Pseudallescheria ellipsoidea F42-3. Their structures were determined by spectroscopic data, ECD calculation, and X-ray single crystal diffraction. The biogenetic pathways of 1-3 were proposed, and 1H-indole-3-carboxylic acid (4), a plausible biosynthetic intermediate, was coisolated.

Discovering new agents active against methicillin-resistant Staphylococcus aureus with ligand-based approaches.

To discover new agents active against methicillin-resistant Staphylococcus aureus (MRSA), in silico models derived from 5451 cell-based anti-MRSA assay data were developed using four machine learning methods, including naïve Bayesian, support vector machine (SVM), recursive partitioning (RP), and k-nearest neighbors (kNN). A total of 876 models have been constructed based on physicochemical descriptors and fingerprints. The overall predictive accuracies of the best models exceeded 80% for both training and test sets. The best model was employed for the virtual screening of anti-MRSA compounds, which were then validated by a cell-based assay using the broth microdilution method with three types of highly resistant MRSA strains (ST239, ST5, and 252). A total of 12 new anti-MRSA agents were confirmed, which had MIC values ranging from 4 to 64 mg/L. This work proves the capacity of combined multiple ligand-based approaches for the discovery of new agents active against MRSA with cell-based assays. We think this work may inspire other lead identification processes when cell-based assay data are available.