Deciphering the Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations.

Thomas Barbot,Liliane Mouawad,Éric Quiniou,Veronica Beswick,Nadège Jamin,Cédric Montigny

doi:10.3389/fmolb.2020.606254

Abstract

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well-understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304, and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

Highlights

The Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA) is a transmembrane protein that transports Ca2+ from the cytoplasm to the sarco/endoplasm, using Adenosine Triphosphate (ATP) as an energy source
SERCA1a is a 110 kDa protein composed of 10 transmembrane helices (TM1-10) and a large cytoplasmic headpiece that consists of three domains: the Nucleotide-binding (N) domain, which binds an ATP molecule, the Phosphorylation (P) domain, which contains the autophosphorylation site and the Actuator (A) domain, which is responsible for the dephosphorylation of the protein (Figure 1A) (MacLennan et al, 1985; Zhang et al, 1998)
Based on Normal Mode calculations and Energy Minimizations, we may summarize the mechanism of action of SLN on SERCA1a as follows

Summary

Introduction

The Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA) is a transmembrane protein that transports Ca2+ from the cytoplasm to the sarco/endoplasm, using ATP as an energy source. The functional cycle may be described in four main steps (Figure 1B, adapted from Møller et al, 2010): I- SERCA1a in the presence of ATP binds two cytoplasmic Ca2+ ions to form the E1.2Ca2+:ATP complex, in which the two Ca2+ binding sites are occluded (PDB ID: 1VFP; Toyoshima and Mizutani, 2004, 3AR2; Toyoshima et al, 2011, 1T5S; Sørensen et al, 2004, and 3TLM; Sacchetto et al, 2012, see Supplementary Table 1 for details about the structures) At this step, two or three protons, H+, are released from the calcium-binding sites into the cytoplasm. Since an ATP analog is always present in the structures that we consider in this study (except for 3W5C which will be discussed below), from here on, ATP will be omitted from the notation of the states

Methods

Results

Discussion

Conclusion