Arabidopsis thaliana HMA2 is a Zn
2+ transporting P
1B-type ATPase required for maintainingplant metal homeostasis. HMA2 and all eukaryote Zn
2+-ATPases have unique conserved N- and C-terminalsequences that diff
erentiate them from other P
1B-type ATPases. Homology modeling and structuralcomparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the
folding present in most P
1B-type ATPase N-terminal metal binding domains (N-MBDs). However,the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence presentin all plant Zn
2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn
2+ (
Kd 0.18
M),Cd
2+ (
Kd 0.27
M), or, with less affinity, Cu
+ (
Kd 13
M). Mutagenesis studies indicate that Cys17,Cys18, and Glu21 participate in Zn
2+ and Cd
2+ coordination, while Cys17 and Glu21, but not Cys18, arerequired for Cu
+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site isunable to bind Zn
2+ or Cd
2+ but it binds Cu
+ with affinity (
Kd 1
M) higher than wild type N-MBD.Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes inmetal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carryingmutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependencesimilar to the wild type enzyme. These observations suggest that plant Zn
2+-ATPase N-MBDs have afolding and function similar to Cu
+-ATPase N-MBDs. However, the unique Zn
2+ coordination via twothiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains.Metal binding through these side chains, although found in diff
erent sequences, appears as a commonfeature of both bacterial and eukaryotic Zn
2+-ATPase N-MBDs.