Mutational analysis of the peptide segment linking phosphorylation and Ca(2+)-binding domains in the sarcoplasmic reticulum Ca(2+)-ATPase.
The sarcoplasmic reticulum ATPase segment extending from the phosphorylation site (Asp-351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6, and M8) retains a marked sequence homology to the corresponding segments of other cation ATPases. We made 26 point mutations in this segment and found that nonconservative mutations of residues that are homologous in various cation ATPases result in strong inhibition of catalytic and transport function. Mutations of nonhomologous residues to match the corresponding residues of other cation ATPases are not inhibitory and, in some cases, produce higher activity. The inhibitory mutations affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+ following enzyme preincubation with EGTA. This suggests that activation of the phosphoryl transfer reaction by Ca2+ binding and vectorial displacement of bound Ca2+ by enzyme phosphorylation do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece through the M4 helix in the membrane-bound region sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.
Zhang
Z
Z
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Sumbilla
C
C
Lewis
D
D
Summers
S
S
Klein
M G
MG
Inesi
G
G
D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D018656:Muscle Fibers, Fast-Twitch; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium