title: Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2.
abstract: Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
authors.forename: Elif
authors.initials: E
authors.lastname: Eren
authors.forename: Manuel
authors.initials: M
authors.lastname: González-Guerrero
authors.forename: Brad M
authors.initials: BM
authors.lastname: Kaufman
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D000251:Adenosine Triphosphatases; C494967:HMA2 protein, Arabidopsis; C109898:Zn(II)-translocating P-type ATPase; D015032:Zinc