Novel Zn2+ Coordination by the Regulatory N-Terminus Metal Binding Domain of Arabidopsis thaliana Zn2+-ATPase HMA2
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- 作者：Elif Eren ; Manuel Gonzá ; lez-Guerrero ; Brad M. Kaufman ; José ; M. Argü ; ello
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：July 3, 2007
- 年：2007
- 卷：46
- 期：26
- 页码：7754 - 7764
- 全文大小：348K
- 年卷期：v.46,no.26(July 3, 2007)
- ISSN：1520-4995
文摘
Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintainingplant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminalsequences that differentiate them from other P1B-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 P1B-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 Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 
ntities/mgr.gif">M),Cd2+ (Kd 0.27 ntities/mgr.gif">M), or, with less affinity, Cu+ (Kd 13 ntities/mgr.gif">M). Mutagenesis studies indicate that Cys17,Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, arerequired for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site isunable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 ntities/mgr.gif">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 Zn2+-ATPase N-MBDs have afolding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ 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 different sequences, appears as a commonfeature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.
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 presentin all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 ntities/mgr.gif">M),Cd2+ (Kd 0.27 ntities/mgr.gif">M), or, with less affinity, Cu+ (Kd 13 ntities/mgr.gif">M). Mutagenesis studies indicate that Cys17,Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, arerequired for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site isunable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 ntities/mgr.gif">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 Zn2+-ATPase N-MBDs have afolding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ 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 different sequences, appears as a commonfeature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.