Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the fast-twitch Ca2+-ATPase.
Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1-E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase.
Adams
P
P
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK. pa2@soton.ac.uk
East
J M
JM
Lee
A G
AG
O'Connor
C D
CD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000378:metabolism; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism; D005813:Genes, Synthetic; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019284:Thapsigargin / Q000378:metabolism
D016227:Benzoquinones; D004791:Enzyme Inhibitors; C067344:2,5-di-tert-butylbenzoquinone; D019284:Thapsigargin; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases