Interaction between the catalytic site and the A-M3 linker stabilizes E2/E2P conformational states of Na+,K +-ATPase

Abstract

The consequences of mutations Ile265 → Ala, Thr 267 → Ala, Gly271 → Ala, and Gly274 → 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 Ile265 → Ala displaces the E1-E2 and E1P-E 2P equilibria in favor of E1/E1P, whereas mutations Thr267 → Ala, Gly271 → Ala, and Gly274 → Ala displace these conformational equilibria in favor of E2/E2P. 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). Destafoilization of E 2/E2P in Ile265 → Ala, as well as a direct effect on the intrinsic affinity of the E2 form for vanadate, may be explained on the basis of the E2 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 Ile265 → Ala, indicating a lack of interference with the catalytic function in E 2/E2P. The effects of mutations Thr267 → Ala, Gly271 → Ala, and Gly274 → Ala provide the first evidence in the literature of a relative stabilization of E 2/E2P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E1/E1P, increased stability of the A3 helix of the A-M3 linker in E2/E2P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.