Metal-Binding Characteristics of the Amino-Terminal Domain of ZntA:  Binding of Lead Is Different Compared to Cadmium and Zinc

Abstract

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).

A Novel Zinc Binding System, ZevAB, Is Critical for Survival of Nontypeable Haemophilus influenzae in a Murine Lung Infection Model

“…Fitting the data to a Langmuir equation yielded an association constant of ZevA for Zn 2ϩ of 1.9 ϫ 10 7 Ϯ 0.3 ϫ 10 7 M Ϫ1 . This affinity is similar to those observed for other known Zn 2ϩ binding proteins (15,24,37). Relative contributions of zevAB and znuABC in zinc utilization.…”

“…The binding affinity of ZevA for Zn 2ϩ was determined using the Zn binding chromophore mag-fura-2 (Invitrogen, Carlsbad, CA) as previously described (18,37,67). Briefly, 10 M ZevA protein and 20 M mag-fura-2 were mixed and titrated with 1 mM Zn 2ϩ .…”

“…, where I is the concentration of mag-fura-2, I free is free mag-fura-2, and K I is the association constant of mag-fura-2 for Zn 2ϩ (K I ϭ 5 ϫ 10 7 M Ϫ1 ) (37,68). The Zn 2ϩ -protein association constant (K a ) and the apparent stoichiometry (n) of ZevA were calculated by fitting the data to the equation…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…Zinc-binding experiments were conducted in the presence of the indicator Mag-Fura-2 (MF) (Molecular Probes, Eugene, OR, USA) according to reported procedures [33][34][35][36]. Absorption spectra were collected from 250 to 800 nm with a Hewlett-Packard 8452A spectrophotometer at ambient temperature.…”

“…The absorbance of apoZnuA was subtracted from each spectrum. Data reduction was performed following reported procedures [33][34][35][36], and data fitting was performed with the program DynaFit [37]. Each spectrum was corrected for dilution and absorbance at 800 nm.…”