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J. Biol. Chem., Vol. 280, Issue 18, 18291-18301, May 6, 2005

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Covalent Cross-links between the Subunit (FXYD2) and and Subunits of Na,K-ATPase

MODELING THE - INTERACTION^*

Maria Füzesi, Kay-Eberhard Gottschalk, Moshit Lindzen, Alla Shainskaya, Bernhard Küster¶||, Haim Garty**, and Steven J. D. Karlish

From the Department of Biological Chemistry and Biological Mass Spectrometry Facility, Weizmann Institute of Science, Rehovoth, 76100, Israel, and the ^¶Protein Interaction Laboratory, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

This study describes specific intramolecular covalent cross-linking of the to and to subunits of pig kidney Na,K-ATPase and rat to co-expressed in HeLa cells. For this purpose pig a and b sequences were determined by cloning and mass spectrometry. Three bifunctional reagents were used: N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), disuccinimidyl tartrate (DST), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide (EDC). NHS-ASA induced -, DST induced - and -, and EDC induced primarily - cross-links. Specific proteolytic and Fe²⁺-catalyzed cleavages located NHS-ASA- and DST-induced - cross-links on the cytoplasmic surface of the subunit, downstream of His²⁸³ and upstream of Val⁴⁴⁰. Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys³⁴⁷ or Lys³⁵² in the S4 stalk segment. Mutational analysis of the rat subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys⁵⁵ and Lys⁵⁶ in the cytoplasmic segment. DST and EDC induced two - cross-links, a major one at the extracellular surface within the segment Gly¹⁴³-Ser³⁰² of the subunit and another within Ala¹-Arg¹⁴². Based on the cross-linking and other data on - proximities, we modeled interactions of the transmembrane -helix and an unstructured cytoplasmic segment SKRLRCGGKKHR of with a homology model of the pig 1 subunit. According to the model, the transmembrane segment fits in a groove between M2, M6, and M9, and the cytoplasmic segment interacts with loops L6/7 and L8/9 and stalk S5.

Received for publication, January 4, 2005 , and in revised form, February 25, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY941203.

^* This work was supported by the Minerva Foundation, the Israel Science Foundation, and the Weizmann Institute renal research fund. 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.

^|| Present address: Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

^** Incumbent of the Hella and Derrick Kleeman chair of biochemistry.

Incumbent of the William Smithburg chair of biochemistry. To whom correspondence should be addressed. Tel.: 972-8-934-2278; Fax: 972-8-934-4118; E-mail: steven.karlish{at}weizmann.ac.il.

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