Interaction between the catalytic site and the A-M3 linker stabilizes E2/E2P conformational states of Na+,K+-ATPase.
The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.
Toustrup-Jensen
Mads
M
Department of Physiology, Institute of Physiology and Biophysics, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark.
Vilsen
Bente
B
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012413:Rubidium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology
D008024:Ligands; D020033:Protein Isoforms; D007532:Isoleucine; D013912:Threonine; D014638:Vanadates; D012965:Sodium Chloride; D010042:Ouabain; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D000409:Alanine; D011188:Potassium; D005998:Glycine