A binding hotspot inTrypanosoma cruzihistidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasiteTrypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure ofT. cruzihistidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind toT. cruziHisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors.T. cruziHisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition ofT. cruziHisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

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Spectroscopic characterization of the heme binding (GAF) domain of two sensor kinases from Methanosarcina acetivorans

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424619500883 ◽

2019 ◽

Vol 23 (07n08) ◽

pp. 930-942

Author(s):

Kerstin Fiege ◽

Christian Twittenhoff ◽

Kathrin Kwiatkowski ◽

Nicole Frankenberg-Dinkel

Keyword(s):

Spectroscopic Characterization ◽

Binding Pocket ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Heme Binding ◽

Methanosarcina Acetivorans ◽

Vis Spectroscopy ◽

Sensor Kinases ◽

Covalently Linked ◽

Heme Cofactor

The sensor kinases MsmS and RdmS from the methanogenic archaeon Methanosarcina acetivorans are multidomain proteins containing a covalently linked heme cofactor. This cofactor is connected via a single cysteine residue in a GAF domain. Although both proteins were shown to display a redox-dependent control of the downstream kinase module, this property appears to be independent of the heme cofactor. We therefore envision an additional sensor role for the heme cofactor. In order to learn more about the heme binding pocket and its constitution, UV-vis spectroscopy in combination with site-directed mutagenesis was performed on the isolated heme-binding sGAF2 domain and the full-length protein. The data indicate a 6-coordinated heme with a proximal histidine ligand and a smaller ligand, likely a water molecule on the distal site. The latter is also thought to be the sensory site and is shown to easily undergo ligand exchange.

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Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

Journal of Molecular Evolution ◽

10.1007/s00239-021-09995-z ◽

2021 ◽

Vol 89 (1-2) ◽

pp. 103-116

Author(s):

Julie Baker Phillips ◽

David H. Ardell

Keyword(s):

Structure Function ◽

Metal Ion ◽

Binding Pocket ◽

Trna Synthetase ◽

Heterologous Gene ◽

Multigene Families ◽

Ion Binding ◽

Trna Genes ◽

Structural Factors ◽

Trna Structure

AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

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Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction: an in silico study

Journal of Computer-Aided Molecular Design ◽

10.1007/s10822-021-00391-9 ◽

2021 ◽

Author(s):

Chiara Luise ◽

Dina Robaa ◽

Wolfgang Sippl

Keyword(s):

Molecular Dynamic ◽

In Silico ◽

Binding Pocket ◽

Binding Mode ◽

Molecular Dynamic Simulations ◽

Flexible Docking ◽

Dynamic Simulations ◽

Binding Mode Prediction ◽

Aromatic Cage ◽

The Right

AbstractSome of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.

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Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317000031 ◽

2017 ◽

Vol 73 (4) ◽

pp. 294-315 ◽

Cited By ~ 12

Author(s):

Kimberly A. Stanek ◽

Jennifer Patterson-West ◽

Peter S. Randolph ◽

Cameron Mura

Keyword(s):

Rna Binding ◽

Binding Pocket ◽

Binding Mode ◽

Evolutionary Conservation ◽

Aquifex Aeolicus ◽

Binding Properties ◽

Bacterial Phyla ◽

Mrna Targets ◽

Small Regulatory Rnas ◽

And Function

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

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Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

mSphere ◽

10.1128/mspheredirect.00340-17 ◽

2017 ◽

Vol 2 (4) ◽

Author(s):

Sanya Chadha ◽

N. Arjunreddy Mallampudi ◽

Debendra K. Mohapatra ◽

Rentala Madhubala

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Essential Gene ◽

Protozoan Parasite ◽

Protein Translation ◽

Trna Synthetase ◽

Parasite Growth ◽

Coding Sequences ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

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Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092912 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C708-C708

Author(s):

Cho Yeow Koh ◽

Jasmine Nguyen ◽

Sayaka Shibata ◽

Zhongsheng Zhang ◽

Ranae Ranade ◽

...

Keyword(s):

Crystal Structures ◽

Trypanosoma Brucei ◽

High Throughput ◽

High Throughput Screening ◽

Protozoan Parasite ◽

Trna Synthetase ◽

Structure Based Drug Design ◽

Chemical Structures ◽

Ic50 Values ◽

Methionyl Trna Synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

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The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode

Journal of Bacteriology ◽

10.1128/jb.185.14.4144-4151.2003 ◽

2003 ◽

Vol 185 (14) ◽

pp. 4144-4151 ◽

Cited By ~ 32

Author(s):

Sheng Ye ◽

Frank von Delft ◽

Alexei Brooun ◽

Mark W. Knuth ◽

Ronald V. Swanson ◽

...

Keyword(s):

Catalytic Domain ◽

Binding Pocket ◽

Binding Mode ◽

Sequence Motif ◽

Binary Complex ◽

Shikimate Dehydrogenase ◽

Conserved Residues ◽

Conserved Sequence ◽

Nicotinamide Mononucleotide ◽

Reversible Reduction

ABSTRACT Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution. The molecule contains two domains, a catalytic domain with a novel open twisted α/β motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

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Pharmacophore Modelling-Based Drug Repurposing Approaches for SARS-CoV-2 Therapeutics

Frontiers in Chemistry ◽

10.3389/fchem.2021.636362 ◽

2021 ◽

Vol 9 ◽

Author(s):

Shailima Rampogu ◽

Keun Woo Lee

Keyword(s):

Effective Treatment ◽

Pharmacophore Model ◽

Drug Repurposing ◽

Binding Pocket ◽

Binding Mode ◽

Computational Method ◽

Dynamics Simulation ◽

Potential Candidate ◽

Pharmacophore Modelling ◽

Discovery Studio

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating effect globally with no effective treatment. The swift strategy to find effective treatment against coronavirus disease 2019 (COVID-19) is to repurpose the approved drugs. In this pursuit, an exhaustive computational method has been used on the DrugBank compounds targeting nsp16/nsp10 complex (PDB code: 6W4H). A structure-based pharmacophore model was generated, and the selected model was escalated to screen DrugBank database, resulting in three compounds. These compounds were subjected to molecular docking studies at the protein-binding pocket employing the CDOCKER module available with the Discovery Studio v18. In order to discover potential candidate compounds, the co-crystallized compound S-adenosyl methionine (SAM) was used as the reference compound. Additionally, the compounds remdesivir and hydroxycholoroquine were employed for comparative docking. The results have shown that the three compounds have demonstrated a higher dock score than the reference compounds and were upgraded to molecular dynamics simulation (MDS) studies. The MDS results demonstrated that the three compounds, framycetin, kanamycin, and tobramycin, are promising candidate compounds. They have represented a stable binding mode at the targets binding pocket with an average protein backbone root mean square deviation below 0.3 nm. Additionally, they have prompted the hydrogen bonds during the entire simulations, inferring that the compounds have occupied the active site firmly. Taken together, our findings propose framycetin, kanamycin, and tobramycin as potent putative inhibitors for COVID-19 therapeutics.

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A Complete Assessment of Dopamine Receptor-Ligand Interactions through Computational Methods

10.20944/preprints201902.0064.v1 ◽

2019 ◽

Author(s):

Beatriz Bueschbell ◽

Carlos A.V. Barreto ◽

Antonio J. Preto ◽

Anke C. Schiedel ◽

Irina S. Moreira

Keyword(s):

Dopamine Receptor ◽

Electrostatic Interactions ◽

Specific Binding ◽

Binding Pocket ◽

Binding Mode ◽

Receptor Subtypes ◽

Receptor Ligand ◽

Pi Stacking ◽

Ligand Interactions ◽

New Treatments

Background: Selectively targeting dopamine receptors has been a persistent challenge in the last years for the development of new treatments to combat the large variety of diseases evolving these receptors. Although, several drugs have been successfully brought to market, the subtype-specific binding mode on a molecular basis has not been fully elucidated. Methods: Homology modeling and molecular dynamics were applied to construct robust conformational models of all dopamine receptor subtypes (D1-like and D2-like receptors). Fifteen structurally diverse ligands were docked to these models. Contacts at the binding pocket were fully described in order to reveal new structural findings responsible for DR sub-type specificity. Results: We showed that the number of conformations for a receptor:ligand complex was associated to unspecific interactions > 2.5 Å and hydrophobic contacts, while the decoys binding energy was influenced by specific electrostatic interactions. Known residues such as 3.32Asp, the serine microdomain and the aromatic microdomain were found interacting in a variety of modes (HB, SB, π-stacking). Purposed TM2-TM3-TM7 microdomain was found to form a hydrophobic network involving Orthosteric Binding Pocket (OBP) and Secondary Binding Pocket (SBP). T-stacking interactions revealed as especially relevant for some large ligands such as apomorphine, risperidone or aripiprazole. Conclusions: This in silico approach was successful in showing known receptor-ligand interactions as well as in determining unique combinations of interactions, key for the design of more specific ligands.

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