Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA

Abstract

The sarcoplasmic reticulum Ca-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Learning of Signaling Networks: Molecular Mechanisms

“…Several proteins display conformational memory, where the protein transiently keeps its active conformation after the dissociation from its former binding partner [13]. Examples include the active state of the endocytosed, unliganded integrin receptor [14], as well as the SERCA Ca-ATP-ase [15]. Here, we propose that conformational memory may participate in the molecular memory formation of single cells (Figure 1).…”

“…--Intrinsically disordered proteins (IDPs): proteins that do not have an ordered 3dimensional structure; in many proteins, structural disorder only extends to a segment (intrinsically disordered region, IDR) of the protein. [13][14][15]. For example, the integrin receptor (β1 subunit) [14], SERCA Ca-ATP-ase [15], and prion-like proteins [3,20,21] all possess conformational memory and participate in cellular learning.…”

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

“…To investigate the PLN effect on ATP-dependent Ca 2+ translocation by SERCA, SSM-based current measurements were carried out on co-reconstituted proteoliposomes containing SERCA and PLN [ 57 ]. The proteoliposomes were adsorbed on the SSM and activated by Ca 2+ and/or ATP concentration jumps.…”

“…The results from pre-steady state charge (calcium) translocation experiments were compared with steady-state measurements of ATPase hydrolytic activity. It was found that the PLN effect on SERCA transport activity depends on substrate conditions, and PLN can establish an inhibitory interaction with multiple conformational states of SERCA (a calcium-free E 2 state, a E 1 -like state promoted by Ca 2+ , and a E 2 -like state promoted by ATP, shown in red in Figure 4 ) with distinct effects on SERCA’s kinetic properties [ 57 ]. It was also noted that once a particular SERCA–PLN inhibitory interaction is established, it remains throughout the SERCA transport and catalytic cycle.…”

Dynamics-driven allostery underlies pre-activation of the regulatory Ca2+-ATPase/phospholamban complex

“…While the application of a variety of simulation and experimental approaches alone has led to a greater insight into the mechanisms for SERCA-PLB regulation, the structural changes induced by Ca 2+ and how those are communicated to couple enzymatic activity with active transport remain poorly defined. More specifically, it remains unknown whether binding of a single Ca 2+ ion initiates SERCA-PLB activation by stabilizing the transport sites [17,22,23] and producing a SERCA structure that is compatible with Ca 2+ binding [24], or by inducing large-scale domain arrangements that produce catalytically competent conformations of SERCA [25,26]. Answering these questions requires knowledge of the structural changes and motions at high spatiotemporal resolution.…”