Multiple metal binding domains enhance the Zn(II) selectivity of the divalent metal ion transporter AztA.
Transition metal-transporting P1B-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We show that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA are functionally nonequivalent, but only with respect to zinc resistance. Inactivation of the a-MBD largely abrogates resistance to high intracellular Zn(II) levels, whereas inactivation of the b-MBD is not as deleterious. In contrast, inactivation of either the a- or b-MBD has little measurable impact on Cd(II) and Pb(II) resistance. The membrane proximal b-MBD binds Zn(II) with a higher affinity than the distal N-terminal a-MBD. Facile Zn(II)-specific intermolecular transfer from the a-MBD to the higher-affinity b-MBD is readily observed by 1H-15N HSQC spectroscopy. Unlike Zn(II), Cd(II) and Pb(II) form saturated 1:1 S4 or S3(O/N) complexes with AztAaHbH, where a single metal ion bridges the two MBDs. We propose that the tandem MBDs enhance Zn(II)-specific transport, while stabilizing a non-native inter-MBD Cd/Pb cross-linked structure that is a poor substrate and/or regulator for the transporter.
Liu
Tong
T
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Reyes-Caballero
Hermes
H
Li
Chenxi
C
Scott
Robert A
RA
Giedroc
David P
DP
D000595:Amino Acid Sequence; D017033:Anabaena / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007854:Lead / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*