Phospholamban domain IB forms an interaction site with the loop between transmembrane helices M6 and M7 of sarco(endo)plasmic reticulum Ca ²⁺ ATPases

Abstract

Transmembrane helix M6 of the sarco(endo)plasmic reticulum Ca 2؉ 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, PLN⌬21-29, 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, PLN⌬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 b5 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. . The phosphorylation and nucleotide binding domains resemble the major and subdomains of the haloacid dehalogenase (4). Unexpected features that are of obvious functional significance are the association of two helices at the N terminus of the molecule with a cytosolic ␤-strand domain between helices M2 and M3 to form an actuator domain and a cytosolic loop (L67) between M6 and M7 (F809-G831), which winds around and links together several elements of the stalk sector. These two features set up linkages that might involve virtually all of the transmembrane helices in the conformational movements that drive Ca 2ϩ transport.The role of amino acid residues in the L67 loop in Ca 2ϩ transport has been investigated through site-directed mutagenesis (5, 6). Mutations K819A and R822A inhibit Ca Phospholamban (PLN) is a 52-aa integral membrane protein, highly expressed in cardiac and slow-twitch skeletal muscle fibers, that interacts with and, at low Ca 2ϩ concentrations, reversibly inhibits the activity of SERCA2a by lowering its apparent affinity for Ca 2ϩ (7). Studies of mice in which PLN is ablated or replaced by highly inhibitory forms of PLN demonstrate that PLN is a modulator of the activity of the Ca 2ϩ pump and a major regulator of the dynamics of cardiac contractility (8-11). It has been proposed that PLN contains three domains. Domain IA, amino acids 1-20, contains sites of regulatory phosphorylation by protein kinase A at S16 and by Ca 2ϩ -calmodulin kinase at T17; domain IB, amino acids 21-30, contains a high proportion of amidated residues; domain II, amino acids 31-52, fo...

Reconstitution of the Cytoplasmic Interaction between Phospholamban and Ca2+-ATPase of Cardiac Sarcoplasmic Reticulum

“…5). These discrepancies might be explained by the involvement of the interaction between the membrane domains and between domain Ib of PLN and the loop between transmembrane helices M6 and M7 of SERCA (Asahi et al, 1999(Asahi et al, , 2001Kimura et al, 1997Kimura et al, , 1998 in the immunoprecipitation study.…”

“…Chemical cross-linking between purified PLN and SERCA2a molecules has provided evidence that PLN directly binds to SERCA2a (James et al, 1989). Studies employing site-specific mutagenesis followed by transient expression in human embryonic kidney 293 cells have revealed that two, or possibly three, interaction sites in the cytoplasmic domains and the membrane helices of both proteins are involved in the inhibitory action of PLN on SERCA2a (Toyofuku et al, 1994a,b;Kimura et al, 1997Kimura et al, , 1998Asahi et al, 1999Asahi et al, , 2001. Based on these observations, we have proposed that PLN and SERCA2a interact via a four (or possibly six)-base circuit through which long range inhibitory interactions are propagated among a series of cytoplasmic and intramembrane interaction sites (MacLennan et al, This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (to Y.K.…”

Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase

“…Indeed, provided SERCA is in a calcium-free state, the interactions between it and PLB remain stable, and dissociation of the two proteins occurs only under conditions of elevated Ca 2ϩ (19). In addition to contacts with M6, further mutational studies have demonstrated an interaction site between PLB domain IB (residues 17-32) and the L67 loop region of Ca 2ϩ ATPase, which links helices M6 and M7 (15). Phosphorylation of PLB may lead to disruption in this region while leaving other interactions intact (15).…”

Spatial and Dynamic Interactions between Phospholamban and the Canine Cardiac Ca2+ Pump Revealed with Use of Heterobifunctional Cross-linking Agents

“…Furthermore, their separation by three residues would place them on the same side of this helix, which would be consistent with their interaction with the same Lys of SERCA2a. This idea contradicts recent models of PLB inter-acting with SERCA2a (18 -20), which assumed an unstructured region between PLB residues 21 and 30, the so-called domain IB (40). Although NMR structures suggest that the transmembrane helix continues without interruption into a helical domain IB (41,42), the collision of this structure with S4 was recently cited as a rationale for unwinding domain IB in a model for the inhibitory interaction with SERCA (20).…”

“…Moreover, it does not seem justified at this time to constrain three-dimensional models of the PLB⅐SERCA2a inhibitory complex (18 -20, 40) with the physical proximity of these particular residues. Although residues 397-402 of SERCA2a are apparently required for functional coupling to PLB (27), this by no means demonstrates that these SERCA residues interact physically with Lys 3 of PLB, as has been often suggested (14,20,40). Likewise, the data provided here and previously (7) provide no evidence for the proposed interaction of residues 27 and 30 of PLB with the loop between M6 and M7 of SERCA (40), an idea that was recently withdrawn (20), and point out the questionable utility of immunoprecipitation for identifying direct residue interactions between PLB and SERCA2a (40) as well as in characterizing thapsigargin effects (12).…”