Protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the SERCA calcium transporter

Abstract

The transport activity of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) in cardiac myocytes is modulated by an inhibitory interaction with a transmembrane peptide, phospholamban (PLB). Previous biochemical studies have revealed that PLB interacts with a specific inhibitory site on SERCA, and low-resolution structural evidence suggests that PLB interacts with distinct alternative sites on SERCA. High-resolution details of the structural determinants of SERCA regulation have been elusive because of the dynamic nature of the regulatory complex. In this study, we used computational approaches to develop a structural model of SERCA–PLB interactions in order to gain a mechanistic understanding of PLB-mediated SERCA transport regulation. We combined steered molecular dynamics (SMD) and membrane protein–protein docking experiments to achieve both a global search and all-atom force calculations to determine the relative affinities of PLB for candidate sites on SERCA. We modeled the binding of PLB to several SERCA conformations, representing different enzymatic states sampled during the calcium transport catalytic cycle. The results of the SMD and docking experiments indicated that the canonical PLB-binding site (comprising transmembrane helices M2, M4, and M9) is the preferred site. This preference was even more stringent for a super-inhibitory PLB variant. Interestingly, PLB-binding specificity became more ambivalent for other SERCA conformers. These results provide evidence for polymorphic PLB interactions with novel sites on M3 and with the outside of the SERCA helix M9. Our findings are compatible with previous physical measurements that suggest that PLB interacts with multiple binding sites, conferring dynamic responsiveness to changing physiological conditions.

Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits

“…However, the crystal structure of the SERCA-PLN complex revealed a PLN dimer bound to SERCA. The PLN pentamer has been shown to bind to an accessory site on SERCA that is distinct from the inhibitory groove and formed by transmembrane segment M3 [35,36]. ).…”

Sarcolambans are phospholamban- and sarcolipin-like regulators of the sarcoplasmic reticulum calcium pump SERCA

“…SERCA binding is not very specific as a variety of nonspecific peptides can also inhibit SERCA (28,29) and non-inhibitory mutants of PLN can still bind (30). Moreover, docking and steered molecular dynamics simulations suggest that PLN can bind in several favorable orientations (31) and that the regulatory interaction is not defined by a single discrete state. While this makes rational design of novel SERCA regulators challenging, the present results provide insight into key structural determinants of regulatory function.…”

Induced fit with replica exchange improves protein complex structure prediction

“…Binding-induced conformational changes, and backbone flexibility at large, has long confounded proteindocking algorithms (47). By improving our understanding of protein interactions and the molecular recognition process, we could determine structures that are yet to be experimentally validated e.g., SERCA-PLB transmembrane complex critical for cardiac function (48), and explore potential association pathways, such as the translocation of protein antibiotics (e.g., colicins) through cellular nutrient transporters (2). Insights into protein docking and binding interfaces have enabled successful computational designs such as symmetrical oligomers for self-assembling nanocages (49,50) and orthogonal designs of cytokine-receptor complexes (51).…”