Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state

Abstract

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+). In the absence of Ca(2+) and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca(2+)), and if millimolar Mg(2+) is present, one Mg(2+) is expected to occupy one of the Ca(2+)-binding sites with a millimolar dissociation constant. This Mg(2+) accelerates the reaction cycle, not permitting phosphorylation without Ca(2+) binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg(2+) state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin, a small regulatory membrane protein of Ca(2+)-ATPase, is bound, stabilizing the E1·Mg(2+) state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca(2+) regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges

“…SR Ca 2+ -ATPase SERCA1a is an integral membrane protein of ∼110 kDa and transports Ca 2+ into the SR lumen against a concentration gradient by hydrolyzing ATP. X-ray crystallography of Ca 2+ -ATPase in different physiological states has revealed that large conformational changes occur during Ca 2+ transport (7,(16)(17)(18)(19). Ca 2+ -ATPase has two high-affinity Ca 2+ -binding sites (I and II, Fig.…”

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.…”

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

“…Phosphorylation from ATP bound mainly to the N-and P-domains in the E1 conformation is triggered by the binding of the two Ca 2ϩ ions in the membrane domain forming the Ca 2 E1 state. Crystal structures indicate that Ca 2ϩ binding to M4 directs a movement of M1-M2 required for bending of the P-domain to form a more compact structure of the ATP site that allows the phosphoryl transfer to the Asp 351 carboxylate group (3,4). The dephosphorylation of E2P occurs as a consequence of the movement of the conserved phosphatase motif of the A-domain, 181 TGES, into close contact with the Asp 351 aspartyl phosphate, allowing the side chain of the glutamate Glu 183 to catalyze a nucleophilic attack of the aspartyl phosphate in a S N 2 reaction, forming a pentacoordinated phosphoryl transition state (5,6).…”