The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm

Abstract

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

“…For instance, the P2A sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) is inhibited by the accessory proteins phospholamban and sarcolipin, small membrane proteins with a single transmembrane segment (4). Sarcolipin uncouples the SERCA pump (5) by trapping it in the Ca 2ϩ -binding conformation (6,7). Autoinhibition by a regulatory domain is observed in a closely related family of P-type ATPases, the P2B calmodulin-activated Ca 2ϩ -ATPases, which occur in both plants and animals (8 -10).…”

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

“…There is a high degree of sequence homology in the transmembrane regions of PLN and SLN, suggesting a similar mode of interaction with SERCA (6,7). Although studies have demonstrated that PLN and SLN use distinct structural elements and mechanisms to regulate SERCA (8,9), crystal structures of the SERCA-SLN (10,11) and SERCA-PLN (12) complexes are remarkably similar to one another.…”

“…In the structure of the SERCA-SLN complex (10,11), Tyr 29 of SLN is proximal to Phe 88 on M2 of SERCA, although Arg 27 and Tyr 31 face away from SERCA. These latter two residues are positioned to protrude into the membrane at the hydrocarbon core-water interface, thereby acting as ballast to properly position SLN in the inhibitory complex (Fig.…”

“…The topology of PLN is organized into three domains as follows: an ␣-helical cytoplasmic domain (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], a linker domain (residues [21][22][23][24][25][26][27][28][29][30], and an ␣-helical transmembrane domain (residues 31-52) (13)(14)(15)(16). The cytoplasmic domain makes a small contribution to SERCA inhibition, yet it has two phosphorylation sites (Ser 16 and Thr 17 ) that play a critical role in the regulation of PLN inhibitory function.…”

Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

“…In agreement with this notion, mutations of an electronegative patch of a CopZ chaperone abolished Cu þ transport in AfCopA (24), which suggests that positively charged amino acids on the MB' helix are the target for chaperone interactions with the surface of the protein core. In the docked position, the chaperone Cu þ -binding site presumably is located close to the putative initial Cu þ receivers Met148, Glu205, and Asp337 (residue numbers refer to LpCopA throughout), as observed for Ca 2þ in the related Ca 2þ -transporting P-type ATPase SERCA1a (9,25). In SERCA1a, the ions are then transferred to two internal Ca 2þ -binding sites (26,27); however, the exact number, location, and chemical nature of the entry pathways in CopA proteins are unknown because the available structural information is limited to E2 states (9,10), where copper already has been deposited on the noncytosolic side.…”