Substituting c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their p38 counterparts affects binding of JNK- and p38-selective inhibitors

2005 ◽  
Vol 438 (2) ◽  
pp. 195-205 ◽  
Author(s):  
Michael Fricker ◽  
Philip LoGrasso ◽  
Semantha Ellis ◽  
Neil Wilkie ◽  
Peter Hunt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Atp Binding ◽  
Amino Acid Residues ◽  
Selective Inhibitors ◽  
Atp Binding Site

scholarly journals Identification of Amino Acid Residues Contributing to the ATP-binding Site of a Purinergic P2X Receptor

2000 ◽  
Vol 275 (44) ◽  
pp. 34190-34196 ◽  
Author(s):  
Lin-Hua Jiang ◽  
François Rassendren ◽  
Annmarie Surprenant ◽  
R. Alan North
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
P2x Receptor ◽  
Atp Binding ◽  
Amino Acid Residues ◽  
Atp Binding Site

scholarly journals Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K+Channel Kir1.1

2002 ◽  
Vol 277 (51) ◽  
pp. 49366-49373 ◽  
Author(s):  
Ke Dong ◽  
LieQi Tang ◽  
Gordon G. MacGregor ◽  
Steven C. Hebert
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Terminal Segment ◽  
K Channel ◽  
Atp Binding ◽  
Amino Acid Residues ◽  
Atp Binding Site ◽  
Arginine Residues

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP2(PI(4,5)P2), regulate the activity of ATP-sensitive K+(KATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP2competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP2antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1CΔC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH2-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP2competed TNP-ATP binding to MBP_1.1CΔC170 with an EC50of 10.9 μm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and KATPchannels and are involved in ATP and/or PIP2effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ΔC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2competition of TNP-ATP (EC50= 5.7 μm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+channels.

scholarly journals Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

2000 ◽  
Vol 351 (3) ◽  
pp. 697-707 ◽  
Author(s):  
Ying-Yi ZHANG ◽  
Tove HAMMARBERG ◽  
Olof RADMARK ◽  
Bengt SAMUELSSON ◽  
Carol F. NG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Tertiary Structure ◽  
Nucleotide Binding ◽  
Peptide Sequencing ◽  
Amino Acid Sequences ◽  
Nucleotide Binding Site ◽  
Atp Binding ◽  
Binding Characteristics ◽  
Atp Binding Site

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5′-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4mol of FSBA/mol of 5LO (of which ATP competed with 1mol/mol) or 0.94mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca2+, which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73–83 (KYWLNDDWYLK, in single-letter amino acid code) and 193–209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.

scholarly journals Binding of ATP to the CBS domains in the C-terminal region of CLC-1

2011 ◽  
Vol 137 (4) ◽  
pp. 357-368 ◽  
Author(s):  
Pang-Yen Tseng ◽  
Wei-Ping Yu ◽  
Hao-Yang Liu ◽  
Xiao-Dong Zhang ◽  
Xiaoqin Zou ◽  
...  
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acids ◽  
Homology Model ◽  
Atp Binding ◽  
Amino Acid Residues ◽  
Atp Binding Site ◽  
Cbs Domain ◽  
C Terminus ◽  
Atp Inhibition ◽  
Cbs Domains

The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1’s C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.

Identification of an Amino Acid in the ATP Binding Site of Na+/K+-ATPase after Photochemical Labeling with 8-Azido-ATP

Biochemistry ◽  
1994 ◽  
Vol 33 (14) ◽  
pp. 4140-4147 ◽  
Author(s):  
Chinh Minh Tran ◽  
Georgios Scheiner-Bobis ◽  
Wilhelm Schoner ◽  
Robert A. Farley
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Atp Binding ◽  
Atp Binding Site

Mutants of protein kinase A that mimic the ATP-binding site of Aurora kinase

2011 ◽  
Vol 440 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Alexander Pflug ◽  
Taianá Maia de Oliveira ◽  
Dirk Bossemeyer ◽  
Richard A. Engh
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Binding Site ◽  
Protein Kinase A ◽  
Aurora Kinase ◽  
Atp Binding ◽  
Atp Binding Site ◽  
High Affinity ◽  
Specific Inhibitors ◽  
Kinase A

We describe in the present paper mutations of the catalytic subunit α of PKA (protein kinase A) that introduce amino acid side chains into the ATP-binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimaeric PKA a valuable tool for structure-based drug discovery tasks. Analysis of crystal structures of the chimaera reveal the roles for individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore, the high affinity for Aurora kinase-specific inhibitors, combined with the favourable crystallizability properties of PKA, allow rapid determination of inhibitor complex structures at an atomic resolution. We demonstrate the utility of the Aurora-chimaeric PKA by measuring binding kinetics for three Aurora kinase-specific inhibitors, and present the X-ray structures of the chimaeric enzyme in complex with VX-680 (MK-0457) and JNJ-7706621 [Aurora kinase/CDK (cyclin-dependent kinase) inhibitor].

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

2019 ◽  
Vol 19 (11) ◽  
pp. 914-926 ◽  
Author(s):  
Maiara Bernardes Marques ◽  
Michael González-Durruthy ◽  
Bruna Félix da Silva Nornberg ◽  
Bruno Rodrigues Oliveira ◽  
Daniela Volcan Almeida ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Binding Site ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Atp Binding ◽  
Human Leukemia ◽  
Atp Binding Site ◽  
Mechanistic Insight ◽  
Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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