Linked Life Data

16051601

Source:http://linkedlifedata.com/resource/pubmed/id/16051601

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
37
pubmed:dateCreated
2005-9-12
pubmed:abstractText
Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
280
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
32349-55
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:16051601-Adenosine Triphosphatases, pubmed-meshheading:16051601-Amino Acid Sequence, pubmed-meshheading:16051601-Amino Acids, pubmed-meshheading:16051601-Animals, pubmed-meshheading:16051601-Binding Sites, pubmed-meshheading:16051601-Crystallography, X-Ray, pubmed-meshheading:16051601-Dose-Response Relationship, Drug, pubmed-meshheading:16051601-Glycosides, pubmed-meshheading:16051601-Hydrogen Bonding, pubmed-meshheading:16051601-Lactones, pubmed-meshheading:16051601-Ligands, pubmed-meshheading:16051601-Macromolecular Substances, pubmed-meshheading:16051601-Models, Molecular, pubmed-meshheading:16051601-Molecular Sequence Data, pubmed-meshheading:16051601-Mutation, pubmed-meshheading:16051601-Ouabain, pubmed-meshheading:16051601-Phosphorylation, pubmed-meshheading:16051601-Protein Binding, pubmed-meshheading:16051601-Protein Conformation, pubmed-meshheading:16051601-Protein Structure, Tertiary, pubmed-meshheading:16051601-Rats, pubmed-meshheading:16051601-Recombinant Fusion Proteins, pubmed-meshheading:16051601-Sequence Homology, Amino Acid, pubmed-meshheading:16051601-Sodium-Potassium-Exchanging ATPase, pubmed-meshheading:16051601-Stomach
pubmed:year
2005
pubmed:articleTitle
Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.
pubmed:affiliation
Department of Biochemistry (160), Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, The Netherlands.
pubmed:publicationType
Journal Article