title: A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2.
abstract: HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-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(1/2) for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 +/- 3 nM). Circular dichroism spectral analysis indicated the presence of 8% alpha-helix, 45% beta-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% alpha-helix, 39% beta-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 P1B-type ATPases, they also appear to regulate enzyme turnover rate.
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: David C
authors.initials: DC
authors.lastname: Kennedy
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Maroney
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
mesh_terms: D015502:Absorptiometry, Photon; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D013438:Sulfhydryl Compounds; D006639:Histidine; D000251:Adenosine Triphosphatases; C494967:HMA2 protein, Arabidopsis; D015032:Zinc; D003545:Cysteine