Crystal structures of the calcium pump and sarcolipin in the [Mg.sup.2+]-bound E1 state

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.sup.2+]-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a [Ca.sup.2+] pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates (1,2). An important outstanding structure is that of the E1 intermediate, which has empty high-affinity [Ca.sup.2+]-binding sites ready to accept new cytosolic [Ca.sup.2+]. In the absence of [Ca.sup.2+] and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for [Ca.sup.2+]) (3), and if millimolar [Mg.sup.2+] is present, one [Mg.sup.2+] is expected to occupy one of the [Ca.sup.2+]-binding sites with a millimolar dissociation constant (4,5). This [Mg.sup.2+] accelerates the reaction cycle (4), not permitting phosphorylation without [Ca.sup.2+] binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1 x [Mg.sup.2+] state at 3.0 [Anstrom] 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 (6), a small regulatory membrane protein of [Ca.sup.2+]-ATPase (7), is bound, stabilizing the E1 x [Mg.sup.2+] state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in [Ca.sup.2+] regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis (11). 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.
New E2 (Supplementary Fig. 1) crystals of SERCAla free from exogenous inhibitors were grown in either 40 mM MgS[O.sub.4] (E2 x S[O.sub.4.sup.2-] crystals) or Mg[Cl.sub.2] (E2 crystals) at pH 6.5 [...]