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J. Biol. Chem., Vol. 278, Issue 46, 46064-46073, November 14, 2003

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Expression of Na⁺,K⁺-ATPase in Pichia pastoris

ANALYSIS OF WILD TYPE AND D369N MUTANT PROTEINS BY FE²⁺-CATALYZED OXIDATIVE CLEAVAGE AND MOLECULAR MODELING^*

David Strugatsky, Kay-Eberhard Gottschalk, Rivka Goldshleger, Eitan Bibi, and Steven J. D. Karlish

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth, 76100, Israel

Na⁺,K⁺-ATPase (pig 1,1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg^-1. Turnover numbers of recombinant and renal Na⁺,K⁺-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe²⁺- and ATP-Fe²⁺-catalyzed oxidative cleavage, described for renal Na⁺,K⁺-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe²⁺ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg²⁺ site in E₂ conformations. The results suggest a possible role of the charge of Asp³⁶⁹ on the E₁ E₂ conformational equilibrium. 5'-Adenylyl-,-imidodi-phosphate(AMP-PNP)-Fe²⁺-catalyzed cleavage of the D369N mutant produces fragments in P (⁷¹²VNDS) and N (near ⁴⁴⁰VAGDA) domains, described for WT, but only at high AMP-PNP-Fe²⁺ concentrations, and a new fragment in the P domain (near ³⁶⁷CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg²⁺ binding to WT and D351N structures of Ca²⁺-ATPase (analogous to Asp³⁶⁹ of Na⁺,K⁺-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn³⁵¹ structure with bound ATP-Mg²⁺ may resemble the transition state of the WT poised for phosphorylation.

Received for publication, July 30, 2003 , and in revised form, August 27, 2003.

^* This work was supported in part by a grant from the Israel Science Foundation 15/00-3. 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.

Supported by a Minerva grant.

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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