PDBsum entry 2kkh

Metal transport

PDB id: 2kkh

Contents

Protein chain

95 a.a. *

Metals

_ZN

Waters ×3

* Residue conservation analysis

PDB id:

2kkh

Name:

Metal transport

Title:

Structure of the zinc binding domain of the atpase hma4

Structure:

Putative heavy metal transporter. Chain: a. Fragment: unp residues 2-96. Engineered: yes

Source:

Arabidopsis thaliana. Mouse-ear cress,thale-cress. Organism_taxid: 3702. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_variant: de3

NMR struc:

20 models

Authors:

M.Zimmerman,O.Clarke,J.M.Gulbis,D.W.Keizer,R.S.Jarvis,C.S.Cobbett, M.G.Hinds,Z.Xiao,A.G.Wedd

Key ref:

M.Zimmermann et al. (2009). Metal binding affinities of Arabidopsis zinc and copper transporters: selectivities match the relative, but not the absolute, affinities of their amino-terminal domains. Biochemistry, 48, 11640-11654. PubMed id: 19883117

Date:

20-Jun-09 Release date: 26-Jan-10

Protein chain

O64474  (HMA4_ARATH) -  Putative cadmium/zinc-transporting ATPase HMA4 from Arabidopsis thaliana

Seq: 1172 a.a.

Struc: 95 a.a.

Enzyme reactions

• Enzyme class 2: E.C.7.2.2.12 - P-type Zn(2+) transporter.
• Reaction: Zn²⁺(in) + ATP + H2O = Zn²⁺(out) + ADP + phosphate + H⁺
• Cofactor: Mg(2+)
• Enzyme class 3: E.C.7.2.2.21 - Cd(2+)-exporting ATPase.
• Reaction: Cd²⁺(in) + ATP + H2O = Cd²⁺(out) + ADP + phosphate + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 48:11640-11654 (2009)

PubMed id: 19883117

Metal binding affinities of Arabidopsis zinc and copper transporters: selectivities match the relative, but not the absolute, affinities of their amino-terminal domains.

M.Zimmermann, O.Clarke, J.M.Gulbis, D.W.Keizer, R.S.Jarvis, C.S.Cobbett, M.G.Hinds, Z.Xiao, A.G.Wedd.

ABSTRACT

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.