Phospholamban domain IB forms an interaction site with the loop between transmembrane helices M6 and M7 of sarco(endo)plasmic reticulum Ca2+ ATPases.
Transmembrane helix M6 of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) has been shown to form a site of interaction with phospholamban (PLN). Site-directed mutagenesis was carried out in the cytoplasmic loop (L67) between M6 and M7 in SERCA1a to detect other SERCA-PLN binding sites. Mutants N810A, D813A, and R822A had diminished ability to interact functionally with PLN, but only D813A and R822A had reduced physical interaction with PLN. PLN mutants R25A, Q26A, N27A, L28A, Q29A, and N30A had enhanced physical interaction with wild-type (wt) SERCA1a, but physical interaction of these PLN mutants with SERCA1a mutants D813A and R822A was reduced about 2.5 fold (range 1.44-2.82). Exceptions were the interactions of PLN N27A and N30A with SERCA1a D813A, which were reduced by 7.3- and 5.8-fold, respectively. A superinhibitory PLN deletion mutant, PLNDelta21-29, had strong physical interactions with SERCA1a and with SERCA1a mutant D813A. Physical interactions with SERCA1a and mutant D813A were sharply diminished, however, for the PLN deletion mutant, PLNDelta21-30, lacking PLN N30. Physical interactions between SERCA1a and a PLN-cytochrome b(5) chimera containing PLN residues 1-29 were much stronger than those between a PLN-cytochrome b(5) chimera containing PLN residues 1-21 and lacking N27. These results suggest that a SERCA1-PLN interaction site occurs between L67 of SERCA1a and domain IB of PLN, which involves SERCA1a D813 and PLN N27 and N30.
Asahi
M
M
Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada.
Green
N M
NM
Kurzydlowski
K
K
Tada
M
M
MacLennan
D H
DH
D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D017384:Sequence Deletion; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C506943:ATP2A1 protein, human