Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites

AbstractThe macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

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

10.1101/078733 ◽

2016 ◽

Author(s):

Kimberly A Stanek ◽

Jennifer P West ◽

Peter S Randolph ◽

Cameron Mura

Keyword(s):

Rna Binding ◽

Binding Pocket ◽

Binding Mode ◽

Evolutionary Conservation ◽

Thermophilic Bacterium ◽

Aquifex Aeolicus ◽

Binding Properties ◽

Bacterial Phyla ◽

Mrna Targets ◽

Small Regulatory Rnas

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-Å resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.AbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) 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 thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 Å resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq 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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A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

10.1101/2020.09.23.309492 ◽

2020 ◽

Author(s):

Veronika Thallmair ◽

Lea Schultz ◽

Siewert J. Marrink ◽

Dominik Oliver ◽

Sebastian Thallmair

Keyword(s):

Binding Site ◽

Md Simulations ◽

Binding Pocket ◽

Binding Mode ◽

Phosphatidyl Serine ◽

Coarse Grained ◽

Membrane Association ◽

Membrane Interface ◽

Binding Properties ◽

Protein Membrane

ABSTRACTPhosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic.Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico.Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

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Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability

Scientific Reports ◽

10.1038/s41598-017-05042-4 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 6

Author(s):

Daniel J. Shaw ◽

Kirsty Robb ◽

Beatrice V. Vetter ◽

Madeline Tong ◽

Virginie Molle ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Binding Pocket ◽

Binding Ability

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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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Techniques to examine nucleotide binding by pseudokinases

Biochemical Society Transactions ◽

10.1042/bst20130075 ◽

2013 ◽

Vol 41 (4) ◽

pp. 975-980 ◽

Cited By ~ 14

Author(s):

Isabelle S. Lucet ◽

Jeffrey J. Babon ◽

James M. Murphy

Keyword(s):

Signal Transduction ◽

Drug Targets ◽

Nucleotide Binding ◽

Biological Functions ◽

Binding Properties ◽

Catalytic Activities ◽

Functional Studies ◽

Signalling Molecules ◽

Advantages And Disadvantages ◽

Human Kinome

Approximately 10% of the human kinome has been classified as pseudokinases due to the absence of one or more of three motifs known to play key roles in the catalytic activities of protein kinases. Structural and functional studies are now emerging, reclassifying this ‘dead’ kinase family as essential signalling molecules that act as crucial modulators of signal transduction. This raises the prospect that pseudokinases may well represent an as-yet-unexplored class of drug targets. However, the extent to which nucleotide binding and catalytic activity contribute to the biological functions of pseudokinases remains an area of great controversy. In the present review, we discuss the advantages and disadvantages of the different methods employed to characterize the nucleotide-binding properties and activity of pseudokinases.

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The C2A-C2B Linker Defines the High Affinity Ca2+ Binding Mode of Rabphilin-3A

Journal of Biological Chemistry ◽

10.1074/jbc.m606746200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 5015-5025 ◽

Cited By ~ 14

Author(s):

Pierre Montaville ◽

Christine Schlicker ◽

Andrei Leonov ◽

Markus Zweckstetter ◽

George M. Sheldrick ◽

...

Keyword(s):

Binding Mode ◽

Bound State ◽

C2 Domain ◽

Binding Assays ◽

Host Proteins ◽

Binding Properties ◽

C2 Domains ◽

Phospholipid Binding ◽

Acidic Residues ◽

Structural Base

The Ca2+ binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca2+ binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca2+-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca2+ ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca2+ binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca2+-bound state of the C2B domain. In addition, this Ca2+ binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca2+ binding mode not only shows how a C2 domain increases its intrinsic Ca2+ affinity, but also provides the structural base for an atypical protein-Ca2+-phospholipid binding mode of rabphilin-3A.

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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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Characterization of the Chimeric PriB-SSBc Protein

International Journal of Molecular Sciences ◽

10.3390/ijms221910854 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10854

Author(s):

En-Shyh Lin ◽

Yen-Hua Huang ◽

Cheng-Yang Huang

Keyword(s):

Replication Fork ◽

Structural Similarity ◽

Binding Properties ◽

Interaction Domain ◽

Ssdna Binding ◽

Initiator Protein ◽

Protein Protein Interaction ◽

Binding Behavior ◽

Oligomerization Domain

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

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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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