Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.
ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.
Okkeri
Juha
J
Institute of Biomedical Sciences/Biochemistry, University of Helsinki, Finland.
Bencomo
Eija
E
Pietilä
Marja
M
Haltia
Tuomas
T
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D006527:Hepatolenticular Degeneration / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D015032:Zinc / Q000378:metabolism*
D027682:Cation Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D000073840:Copper-Transporting ATPases; D015032:Zinc