Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity.
ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ions. Two proteins, deltaN-ZntA, in which the N-terminal domain is deleted, and C59A/C62A-ZntA, in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can only bind one metal ion. Because C59A/C62A-ZntA can bind a metal ion at the transmembrane site, the N-terminal domain does not block direct access of metal ions to it from the cytosol. A third mutant protein, C392A/C394A-ZntA, in which cysteines from the conserved CPC motif in transmembrane helix 6 are altered, binds metal ions only at the N-terminal site, indicating that both these cysteines form part of the transmembrane site. The metal affinity of the transmembrane site was determined in deltaN-ZntA and C59A/C62A-ZntA by competition titration using a metal ion indicator and by tryptophan fluorescence quenching. The binding affinity for the physiological substrates, Zn2+, Pb2+, and Cd2+, as well as for the extremely poor substrates, Cu2+, Ni2+, and Co2+, range from 10(6)-10(10) M(-1), and does not correlate with the metal selectivity shown by ZntA. Selectivity in ZntA possibly results from differences in metal-binding geometry that produce different structural responses. The affinity of the transmembrane site for metal ions is of similar magnitude to that of the N-terminal site [Liu J. et al. (2005) Biochemistry 44, 5159-5167]; thus, metal transfer between them would be facile.
Liu
Junbo
J
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Dutta
Sabari J
SJ
Stemmler
Ann J
AJ
Mitra
Bharati
B
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D013379:Substrate Specificity
D008565:Membrane Proteins; D008670:Metals; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase