A hallmark of phospholamban functional divergence is located in the N-terminal phosphorylation domain

Eli Fernández-de Gortari; Rodrigo Aguayo-Ortiz; Joseph M Autry; L Michel Espinoza-Fonseca

doi:10.1016/j.csbj.2020.02.016

A hallmark of phospholamban functional divergence is located in the N-terminal phosphorylation domain

Comput Struct Biotechnol J. 2020 Feb 28:18:705-713. doi: 10.1016/j.csbj.2020.02.016. eCollection 2020.

Authors

Eli Fernández-de Gortari¹, Rodrigo Aguayo-Ortiz¹, Joseph M Autry^{2

3}, L Michel Espinoza-Fonseca¹

Affiliations

¹ Center for Arrhythmia Research, Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
² Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
³ Biophysical Technology Center, University of Minnesota, Minneapolis, MN 55455, USA.

Abstract

Sarcoplasmic reticulum Ca²⁺ pump (SERCA) is a critical component of the Ca²⁺ transport machinery in myocytes. There is clear evidence for regulation of SERCA activity by PLB, whose activity is modulated by phosphorylation of its N-terminal domain (residues 1-25), but there is less clear evidence for the role of this domain in PLB's functional divergence. It is widely accepted that only sarcolipin (SLN), a protein that shares substantial homology with PLB, uncouples SERCA Ca²⁺ transport from ATP hydrolysis by inducing a structural change of its energy-transduction domain; yet, experimental evidence shows that the transmembrane domain of PLB (residues 26-52, PLB_26-52) partially uncouples SERCA in vitro. These apparently conflicting mechanisms suggest that PLB's uncoupling activity is encoded in its transmembrane domain, and that it is controlled by the N-terminal phosphorylation domain. To test this hypothesis, we performed molecular dynamics simulations (MDS) of the binary complex between PLB_26-52 and SERCA. Comparison between PLB_26-52 and wild-type PLB (PLB_WT) showed no significant changes in the stability and orientation of the transmembrane helix, indicating that PLB_26-52 forms a native-like complex with SERCA. MDS showed that PLB_26-52 produces key intermolecular contacts and structural changes required for inhibition, in agreement with studies showing that PLB_26-52 inhibits SERCA. However, deletion of the N-terminal phosphorylation domain facilitates an order-to-disorder shift in the energy-transduction domain associated with uncoupling of SERCA, albeit weaker than that induced by SLN. This mechanistic evidence reveals that the N-terminal phosphorylation domain of PLB is a primary contributor to the functional divergence among homologous SERCA regulators.

Keywords: Calcium pump; Functional divergence; M4S4, cytosolic extension of transmembrane helix M4; Molecular dynamics simulations; PLB, phospholamban; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Phospholamban; Phosphorylation domain; RMSD, root mean square deviation; SERCA, sarcoplasmic reticulum Ca2+-ATPase; SLN, sarcolipin; Sarcolipin.

Grants and funding

R01 GM120142/GM/NIGMS NIH HHS/United States