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 Call ATPase (SER CA) has been shown to form a site of interaction with phospholamban (PLN). Site-directed mutagenesis was carried out in the cytoplasmic loop (L67) bet ween M6 and M7 in SERCA1a to detect other SERCA-PLN binding sites. Mutants N810A, D813A, and R822A had diminished ability to interact functionally wit h Pill but only D813A and R822A had reduced physical interaction with PLN. PLN mutants R25A, Q26A, N27A, L28A, Q29A, and N30A had enhanced physical in teraction with wild-type (wt) SERCA1a, but physical interaction of these PL N 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 wit h SERCA1a D813A, which were reduced by 7.3- and 5.8-fold, respectively. A s uperinhibitory PLN deletion mutant, PLN Delta 21-29, had strong physical in teractions with SERCA1a and with SERCA1a mutant D813A. Physical interaction s with SERCA1a and mutant D813A were sharply diminished, however, for the P LN deletion mutant, PLN Delta 21-30, 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 bs chimera cont aining PLN residues 1-21 and lacking N27. These results suggest that a SERC A1-PLN interaction site occurs between L67 of SERCA1a and domain IB of PLN, which involves SERCA1a D813 and PLN N27 and N30.