Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc.
ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).
Liu
Junbo
J
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Stemmler
Ann J
AJ
Fatima
Juhi
J
Mitra
Bharati
B
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002104:Cadmium / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D007700:Kinetics; D007854:Lead / Q000378:metabolism; D013058:Mass Spectrometry; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D004269:DNA, Bacterial; D008670:Metals; D011994:Recombinant Proteins; D002104:Cadmium; D007854:Lead; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D015032:Zinc