Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 284, Issue 18, Pages 12258-12265Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M900977200
Keywords
-
Categories
Funding
- Danish Medical Research Council
- Novo Nordisk Foundation
- Lundbeck Foundation
- Danish National Research Foundation-PUMPKIN
Ask authors/readers for more resources
Ion translocation by the sarcoplasmic reticulum Ca2+-ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 (A-M3 linker) by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca2+ and ATP and to form the phosphorylated Ca(2)E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an alpha-helix (A3 helix) in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca(2)E1P state and thereby to drive the transition to E2P.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available