Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli.
ATP7B, the Wilson disease-associated Cu(I)-transporter, and ZntA from Escherichia coli are soft metal P1-type ATPases with mutually exclusive metal ion substrates. P1-type ATPases have a distinctive amino-terminal domain containing the conserved metal-binding motif GXXCXXC. ZntA has one copy of this motif while ATP7B has six copies. The effect of interchanging the amino-terminal domains of ATP7B and ZntA was investigated. Chimeric proteins were constructed in which either the entire amino-terminal domain of ATP7B or only its sixth metal-binding motif replaced the amino-terminal domain of ZntA. Both chimeras conferred resistance to lead, zinc, and cadmium salts but not to copper salts. The purified chimeras displayed activity with lead, cadmium, zinc, and mercury, which are substrates of ZntA. There was no activity with copper or silver, which are substrates of ATP7B. The chimeras were 2-3-fold less active than ZntA. Thus, the amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. Also, these results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity.
Hou
Z J
ZJ
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Narindrasorasak
S
S
Bhushan
B
B
Sarkar
B
B
Mitra
B
B
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D000073840:Copper-Transporting ATPases; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D006527:Hepatolenticular Degeneration / Q000201:enzymology*; D007854:Lead / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D027682:Cation Transport Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D007854:Lead; D003300:Copper; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D000073840:Copper-Transporting ATPases; D015032:Zinc