scholarly journals Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling

2019 ◽  
Vol 12 (580) ◽  
pp. eaav9504 ◽  
Author(s):  
Tristan Wagner ◽  
Gwénaëlle André-Leroux ◽  
Valérie Hindie ◽  
Nathalie Barilone ◽  
María-Natalia Lisa ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Central Element ◽  
Binding Pocket ◽  
Signaling Cascades ◽  
Binding Modes ◽  
Allosteric Site ◽  
Fha Domain ◽  
Protein Partners ◽  
Intramolecular Association ◽  
Structural Insights

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

scholarly journals Recent Advances in the Discovery of CK2 Allosteric Inhibitors: From Traditional Screening to Structure-Based Design

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 870 ◽  
Author(s):  
Xiaolan Chen ◽  
Chunqiong Li ◽  
Dada Wang ◽  
Yu Chen ◽  
Na Zhang
Keyword(s):  
Protein Kinase Ck2 ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Modes ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Atp Binding Site ◽  
Recent Advances ◽  
Allosteric Sites ◽  
Kinase Ck2

Protein kinase (CK2) has emerged as an attractive cancer therapeutic target and recent efforts have been made to develop its inhibitors. However, the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket (orthosteric site) of kinase family. As an alternative strategy, allosteric inhibitors, by targeting the much more diversified allosteric site relative to the conserved ATP-binding site, achieve better pharmacological advantages than orthosteric inhibitors. Traditional serendipitous screening and structure-based design are robust tools for the discovery of CK2 allosteric inhibitors. In this review, we summarize the recent advances in the identification of CK2 allosteric inhibitors. Firstly, we briefly present the CK2 allosteric sites. Then, the allosteric inhibitors targeting the well-elucidated allosteric sites (α/β interface, αD pocket and interface between the Glycine-rich loop and αC-helix) are highlighted in the discovery process and possible binding modes with the allosteric sites are described. This study is expected to provide valuable clues for the design of CK2 allosteric inhibitors.

Application of Molecular Docking for the Development of Improved HIV-1 Reverse Transcriptase Inhibitors

2020 ◽  
Vol 16 ◽  
Author(s):  
Arash Soltani ◽  
Seyed Isaac Hashemy ◽  
Farnaz Zahedi Avval ◽  
Houshang Rafatpanah ◽  
Seyed Abdolrahim Rezaee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Capacity ◽  
Binding Pocket ◽  
Ligand Docking ◽  
Binding Modes ◽  
Wild Type ◽  
Parent Molecule ◽  
Discovery Studio ◽  
Approved Drugs ◽  
Hiv 1

Introoduction: Inhibition of the reverse transcriptase (RT) enzyme of human immunodeficiency virus (HIV) by low molecular weight inhibitors is still an active area of research. Here, protein-ligand interactions and possible binding modes of novel compounds with the HIV-1 RT binding pocket (the wild-type as well as Y181C and K103N mutants) were obtained and discussed. Methods: A molecular fragment-based approach using FDA-approved drugs were followed to design novel chemical derivatives using delavirdine, efavirenz, etravirine and rilpivirine as the scaffolds. The drug-likeliness of the derivatives was evaluated using Swiss-ADME. Then the parent molecule and derivatives were docked into the binding pocket of related crystal structures (PDB ID: 4G1Q, 1IKW, 1KLM and 3MEC). Genetic Optimization for Ligand Docking (GOLD) Suite 5.2.2 software was used for docking and the results analyzed in the Discovery Studio Visualizer 4. A derivative was chosen for further analysis, if it passed drug-likeliness and the docked energy was more favorable than that of its parent molecule. Out of the fifty-seven derivatives, forty-eight failed in druglikeness screening by Swiss-ADME or in docking stage. Results: The final results showed that the selected compounds had higher predicted binding affinities than their parent scaffolds in both wild-type and the mutants. Binding energy improvement was higher for the structures designed based on second-generation NNRTIs (etravirine and rilpivirine) than the first-generation NNRTIs (delavirdine and efavirenz). For example, while the docked energy for rilpivirine was -51 KJ/mol, it was improved for its derivatives RPV01 and RPV15 up to -58.3 and -54.5 KJ/mol, respectively. Conclusion: In this study, we have identified and proposed some novel molecules with improved binding capacity for HIV RT using fragment-based approach.

scholarly journals Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1

2010 ◽  
Vol 84 (10) ◽  
pp. 5391-5403 ◽  
Author(s):  
Einari A. Niskanen ◽  
Teemu O. Ihalainen ◽  
Olli Kalliolinna ◽  
Milla M. Häkkinen ◽  
Maija Vihinen-Ranta
Keyword(s):  
Binding Pocket ◽  
Canine Parvovirus ◽  
Dominant Negative ◽  
Atp Binding ◽  
Helicase Domain ◽  
Genome Replication ◽  
Binding Modes ◽  
Ns1 Protein ◽  
Sensory Element ◽  
Arginine Finger

ABSTRACT The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruses. When provided in trans, all except the R508A mutation reduced infectivity in a dominant-negative manner, possibly by hindering genome replication. These results suggest that the conserved R510 residue, but not R508, is the arginine finger sensory element of CPV NS1. Moreover, fluorescence recovery after photobleaching (FRAP), complemented by computer simulations, was used to assess the binding properties of mutated fluorescent fusion proteins. These experiments identified ATP-dependent and -independent binding modes for NS1 in living cells. Only the K406M mutant had a single binding site, which was concluded to indicate ATP-independent binding. Furthermore, our data suggest that DNA binding of NS1 is dependent on its ability to both bind and hydrolyze ATP.

scholarly journals Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Akira Karasawa ◽  
Toshimitsu Kawate
Keyword(s):  
P2x7 Receptor ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Atp Binding ◽  
Allosteric Site ◽  
Channel Opening ◽  
A Site ◽  
Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

scholarly journals Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

2009 ◽  
Vol 106 (37) ◽  
pp. 15616-15621 ◽  
Author(s):  
Masataka Umitsu ◽  
Hiroshi Nishimasu ◽  
Akiko Noma ◽  
Tsutomu Suzuki ◽  
Ryuichiro Ishitani ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Binding Pocket ◽  
Structural Basis ◽  
Binding Modes ◽  
Substrate Binding Pocket ◽  
Methyl Group ◽  
Group Transfer ◽  
Canonical Class ◽  
Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

Structural insights into the multiple binding modes of Dimethyl Fumarate (DMF) and its analogs to the Kelch domain of Keap1

FEBS Journal ◽  
2020 ◽  
Author(s):  
Sruthi Unni ◽  
Prashant Deshmukh ◽  
Gopinatha Krishnappa ◽  
Padmini Kommu ◽  
Balasundaram Padmanabhan
Keyword(s):  
Dimethyl Fumarate ◽  
Binding Modes ◽  
Multiple Binding ◽  
Kelch Domain ◽  
Structural Insights

scholarly journals An arrestin-1 surface opposite of its interface with photoactivated rhodopsin engages with enolase-1

2020 ◽  
Vol 295 (19) ◽  
pp. 6498-6508
Author(s):  
Connie Jaqueline Miranda ◽  
Nicole Fernandez ◽  
Nader Kamel ◽  
Daniel Turner ◽  
Del Benzenhafer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Model ◽  
Signaling Cascades ◽  
Contact Sites ◽  
Binding Partners ◽  
Protein Partners ◽  
Fluorescence Quench ◽  
Selective Substitution ◽  
Additional Protein ◽  
G Protein Coupled

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

scholarly journals PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response

2018 ◽  
Vol 115 (21) ◽  
pp. E4861-E4869 ◽  
Author(s):  
Khaled A. Selim ◽  
Florian Haase ◽  
Marcus D. Hartmann ◽  
Martin Hagemann ◽  
Karl Forchhammer
Keyword(s):  
Inorganic Carbon ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Oxygenic Photosynthesis ◽  
Binding Modes ◽  
Signaling Protein ◽  
Evolutionarily Conserved ◽  
Photosynthetic Production ◽  
The Individual ◽  
Physiological And Biochemical

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO2 concentrations, they evolved a CO2-concentrating mechanism (CCM) to augment intracellular inorganic carbon (Ci) levels for efficient CO2 fixation. However, how cyanobacteria sense the fluctuation in Ci is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique PII-like signaling protein, which provides new insights into Ci sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical PII proteins. This clearly indicates that proteins of the PII superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in Ci acclimation. Collectively, our results suggest that SbtB acts as a Ci sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

scholarly journals An allosteric site on MKP5 reveals a strategy for small-molecule inhibition

2020 ◽  
Vol 13 (646) ◽  
pp. eaba3043 ◽  
Author(s):  
Zachary T. K. Gannam ◽  
Kisuk Min ◽  
Shanelle R. Shillingford ◽  
Lei Zhang ◽  
James Herrington ◽  
...  
Keyword(s):  
Small Molecule ◽  
Binding Pocket ◽  
Muscle Disease ◽  
Mitogen Activated Protein Kinase ◽  
Allosteric Site ◽  
Dystrophic Muscle ◽  
Smad2 Phosphorylation ◽  
Mitogen Activated Protein ◽  
P38α Mapk ◽  
Tgf Β1

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered “undruggable,” but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK–derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-β1 signaling in muscle and that the inhibitor blocked TGF-β1–mediated Smad2 phosphorylation. TGF-β1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

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