Identifying Calcium ion Access Points and Transport Pathways in SERCA

Abstract

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is one of the best structurally characterized membrane transporters with numerous high resolution crystal structures available for several different functional states. These structures have clearly established the involvement of large conformational changes in transporting Ca2+ ions across the sarcoplasmic reticulum membrane against the concentration gradient, harvesting the required energy from ATP-hydrolysis. Unfortunately, it still has not been possible to crystallize SERCA (or any other P-type ATPase) in a conformation with an open cytoplasmic pathway leading to the two Ca2+ binding sites located in the middle of the transmembrane domain, halfway across the bilayer. Different Ca2+ entry points have been suggested, however, how Ca2+ reach the binding sites is yet to be learned.We have studied different functional states of SERCA embedded in a lipid bilayer and surrounded by water molecules and potassium chloride with extensive all-atom molecular dynamics simulations. Simulations of Ca2+-free states demonstrate how positive ions are attracted to the cytoplasmic surface of the transmembrane domain at a region close to the kinked part of transmembrane helix 1 (TM1). The kinked part is rich in acidic residues. Ion densities show that potassium ions accumulate in this region, and electrostatic potential maps calculated for the protein likewise identifies a negative potential here. Simulations of Ca2+-bound states, with occluded Ca2+ binding sites, illustrates water molecules actually finding their way into the ion binding site nearest the cytoplasmic surface. This water pathway begins from the region close to the TM1-kink seen to attract positive charge in Ca2+-free states.On this basis, we suggest that at least one Ca2+ ion, and probably both, access the Ca2+ ion binding sites through a pathway starting close the TM1-kink, which with several acidic residues contributes to the attraction of ions.