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J. Biol. Chem., Vol. 283, Issue 41, 27850-27858, October 10, 2008

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The Fourth Extracellular Loop of the Subunit of Na,K-ATPase

FUNCTIONAL EVIDENCE FOR CLOSE PROXIMITY WITH THE SECOND EXTRACELLULAR LOOP^*

Oihana Capendeguy¹, Justyna Iwaszkiewicz¹, Olivier Michielin^¶||, and Jean-Daniel Horisberger²

From the Department of Pharmacology and Toxicology, the Swiss Institute of Bioinformatics, the ^¶Ludwig Institute for Cancer Research/Lausanne Branch, and the ^||Centre Pluridisciplinaire d'Oncologie, University of Lausanne, 1005 Lausanne, Switzerland

Na,K-ATPase is the main active transport system that maintains the large gradients of Na⁺ and K⁺ across the plasma membrane of animal cells. The crystal structure of a K⁺-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr³⁰⁸ and Asp⁸⁸⁴ residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the β subunit, can be exposed to the exterior of the protein and can easily interact with the β subunit.

Received for publication, March 19, 2008 , and in revised form, July 21, 2008.

^* This work was supported by Swiss National Fund Grant 31-65441.01 (to J.-D. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. de Pharmacologie et de Toxicologie, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Tel.: 41-21-692-5362; Fax: 41-21-692-5355; E-mail: Jean-Daniel.Horisberger{at}unil.ch.

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