The length of the A-M3 linker is a crucial determinant of the rate of the Ca2+ transport cycle of sarcoplasmic reticulum Ca2+-ATPase.
Ion translocation by the sarcoplasmic reticulum Ca(2+)-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 Ca(2+) 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.
Holdensen
Anne Nyholm
AN
Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Physiology and Biophysics, Aarhus University, DK-8000 Aarhus C, Denmark.
Andersen
Jens Peter
JP
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D004795:Enzyme Stability / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics
D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases