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

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

scholarly journals Tubulin-nucleotide interactions. Effects of removal of exchangeable guanine nucleotide on protein conformation and microtubule assembly

1987 ◽  
Vol 241 (1) ◽  
pp. 105-110 ◽  
Author(s):  
E J Manser ◽  
P M Bayley
Keyword(s):  
Alkaline Phosphatase ◽  
Binding Site ◽  
Protein Interactions ◽  
Tertiary Structure ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Microtubule Assembly ◽  
Structural Requirement ◽  
Tubulin Dimer ◽  
Microtubule Protein

The removal of tightly bound GDP from the exchangeable nucleotide-binding site of tubulin has been performed with alkaline phosphatase under conditions which essentially retain the assembly properties of the protein. When microtubule protein is treated with alkaline phosphatase, nucleotide is selectively removed from tubulin dimer rather than from MAP (microtubule-associated protein)-containing oligomeric species. Tubulin devoid of E-site (the exchangeable nucleotide-binding site of the tubulin dimer) nucleotide shows enhanced proteolytic susceptibility of the beta-subunit to thermolysin and decreased protein stability, consistent with nucleotide removal causing changes in protein tertiary structure. Pyrophosphate ion (3 mM) is able to promote formation of normal microtubules in the complete absence of GTP by incubation at 37 degrees C either with nucleotide-depleted microtubule protein or with nucleotide-depleted tubulin dimer to which MAPs have been added. The resulting microtubules contain up to 80% of tubulin lacking E-site nucleotide. In addition to its effects on nucleation, pyrophosphate competes weakly with GDP bound at the E-site. It is deduced that binding of pyrophosphate at a vacant E-site can promote microtubule assembly. The minimum structural requirement for ligands to induce tubulin assembly apparently involves charge neutralization at the E-site by bidentate ligation, which stabilizes protein domains in a favourable orientation for promoting the supramolecular protein-protein interactions involved in microtubule formation.

scholarly journals The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP

2004 ◽  
Vol 377 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Juha OKKERI ◽  
Liisa LAAKKONEN ◽  
Tuomas HALTIA
Keyword(s):  
Escherichia Coli ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Molecular Model ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Disease Mutations ◽  
Disease Protein ◽  
P Type

In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain. The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx. 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase. None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA. However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases. This motif is also found in Wilson disease protein where several disease mutations cluster in it. In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503→Ser), G505R and A508F of ZntA. At low [ATP], these mutant ATPases are poorly phosphorylated. The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher [ATP], suggesting that these mutations lower the affinity for ATP. In all three mutant ATPases, phosphorylation by Pi has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding. In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template. In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site. In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation.

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 ATP binding to bovine heart cytochrome c oxidase. A photoaffinity labelling study

1986 ◽  
Vol 234 (1) ◽  
pp. 241-243 ◽  
Author(s):  
C Montecucco ◽  
G Schiavo ◽  
R Bisson
Keyword(s):  
Cytochrome C ◽  
Binding Site ◽  
Cytochrome C Oxidase ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Regulatory Role ◽  
Atp Binding ◽  
Kinetic Properties ◽  
Bovine Heart ◽  
Enzyme Subunits

ATP influences the kinetic properties of cytochrome c oxidase. A photoactivatable radioactive ATP analogue was used to localize the nucleotide-binding site on the bovine heart enzyme. Subunits IV and VIII were specifically labelled, suggesting that these two nuclear-coded polypeptides may play a regulatory role on the oxidase functions.

scholarly journals Exploring conformational equilibria of a heterodimeric ABC transporter

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
M Hadi Timachi ◽  
Cedric AJ Hutter ◽  
Michael Hohl ◽  
Tufa Assafa ◽  
Simon Böhm ◽  
...  
Keyword(s):  
Binding Site ◽  
High Temperatures ◽  
Abc Transporter ◽  
Thermotoga Maritima ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Molecular Feature ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Conformational Equilibria

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.

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].

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