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J. Biol. Chem., Vol. 281, Issue 45, 33881-33891, November 10, 2006

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A Novel Regulatory Metal Binding Domain Is Present in the C Terminus of Arabidopsis Zn²⁺-ATPase HMA2^*

Elif Eren, David C. Kennedy, Michael J. Maroney, and José M. Argüello¹

From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 and the Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003

HMA2 is a Zn²⁺-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn²⁺ efflux. Distinct from P_1B-type ATPases, plant Zn²⁺-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn²⁺ K for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn²⁺ with high affinity (K_d = 16 ± 3nM). Circular dichroism spectral analysis indicated the presence of 8% -helix, 45% -sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% -helix, 39% -sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn²⁺ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn²⁺ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn²⁺ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn²⁺ binding to the C terminus, pointing out the importance of His residues in Zn²⁺ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn²⁺ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)₄ coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn²⁺-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P_1B-type ATPases, they also appear to regulate enzyme turnover rate.

Received for publication, May 31, 2006 , and in revised form, September 13, 2006.

^* This work was supported by National Science Foundation Grant MCM-0235165 (to J. M. A.) and National Institutes of Health Grant R01-GM061696 (to M. J. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3, two equations, and data analysis.

¹ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Worcester Polytechnic Inst., 100 Institute Rd, Worcester, MA 01609. Tel.: 508-831-5326; Fax: 508-831-5933; E-mail: arguello{at}wpi.edu.

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