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.

Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum is the beststudied member of the P-type or E1/E2 type ion transporting ATPases. It has been crystallized in seven different states that cover nearly the entire reaction cycle. crystal structure ͉ phosphorylation ͉ P-type ATPase C a 2ϩ -ATPase from skeletal muscle sarcoplasmic reticulum (SERCA1a) is an ATP-powered calcium pump that transfers Ca 2ϩ from the cytoplasm to the lumen of sarcoplasmic reticulum against a Ͼ10 4 concentration gradient. It is an integral membrane protein of M r 110K (1), and it pumps ions by alternating the affinity of the transmembrane (TM) binding sites and synchronizing opening and closing of the cytoplasmic and luminal gates. According to the conventional E1-E2 theory (2-4), E1 and E2 respectively refer to high-affinity and low-affinity states to Ca 2ϩ . Gating of the ion pathway is coupled to autophosphorylation and dephosphorylation of the ATPase. Phosphoryl transfer from ATP to an Asp in the cytoplasmic domain (i.e., E1⅐2Ca 2ϩ 3 E1P; here P stands for phosphorylated state) closes the cytoplasmic gate, and the release of ADP triggers a change in affinity of the Ca 2ϩ binding sites (i.e., E1P 3 E2P) and opening of the luminal gate. Hydrolysis of the aspartylphosphate (E2P 3 E2) closes the gate.

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca 2þ -ATPase, a representative P-type ATPase, in the absence of Ca 2þ with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca 2þ -ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca 2þ -ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.crystallography | ion pump | nucleotide derivatives T rinitrophenyl (TNP)-nucleotides (1) are often used for probing the structure of ATP-binding sites and conformational changes arising from nucleotide binding (2, 3), and for measuring the affinity of ATP by competition experiments (2, 4). It is a preferred ATP analogue for photochemical crosslinking with azide derivatives (5). These applications utilize the enhancement of fluorescence or absorption of visible light of the TNP group upon binding to a protein (6). Because of its sensitivity, competition with ATP/ADP has been a valuable means for examining mutational effects on nucleotide affinity (7). Thus, TNP nucleotides have been widely used with F1 (8), myosin (1), and P-type ATPases (2-5, 7, 9, 10), among others.Nonetheless, whether TNP derivatives are good mimics of authentic adenine nucleotides (AxPs) may be questionable. In several proteins TNP nucleotides have much higher affinities than the genuine AxPs. For instance, TNP-ATP is a high affinity (nM) antagonist of P2X receptors, which have IC 50 for ATP (or AMPPCP) in the μM range (11). The affinity is at least one order of magnitude higher in the E2 states of Ca 2þ -ATPase (3, 12) and Na þ , K þ -ATPase (4), representative P-type ATPases. Furthermore, TNP-AMP binds to Ca 2þ -ATPase similarly to or even more strongly than TNP-ATP (12), in marked contrast to AxPs. Thus, a substantially different binding mode of TNP derivatives is suggested. Although more than 20 entries are registered in the Protein Data Bank (PDB) for Ca 2þ -ATPase (reviewed in ref. 13), no structure with a bound TNP nucleotide exists. In fact, only three crystal structures have been published with bound TNP nucleotides. They are a bacterial h...

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A new method of X-ray solvent contrast modulation was developed to visualize lipid bilayers in crystals of membrane proteins at a high enough resolution to resolve individual phospholipids molecules (~3.5 Å ). Visualization of lipid bilayer has been escaping from conventional crystallographic methods due to its extreme flexibility, and our knowledge on the behavior of lipid bilayer is still very much limited. Here we applied the new method of X-ray solvent contrast modulation to crystals of Ca2+-ATPase in 4 different physiological states. As phospholipids have to be added to make crystals of Ca2+-ATPase, it is expected that lipid bilayers are present in the crystals. Moreover, transmembrane helices of Ca2+-ATPase rearrange drastically during the reaction cycle and some of them show substantial movements perpendicular to the bilayer plane. Thus these crystals provide a rare opportunity to directly visualize phospholipids interacting with a membrane protein in different conformations. Complete diffraction data covering from 200 to 3.2 Å resolution were collected at BL41XU, Spring-8, using an R-Axis V imaging plate detector for crystals soaked in solvent of different electron density. A new concept "solvent exchange probability", which should be 1 in the bulk solvent, 0 inside the protein and an intermediate at interface, was introduced and used as a restraint for real space phase improvement. The electron density maps thus obtained clearly show that: (i) Phospholipid molecules surrounding the protein are fixed apparently by Arg/Lys-phosphate salt bridges or Trp-carbonyl hydrogen bonds and follow the movements of transmembrane helices. Movements of as large as 12 Å are allowed. (ii) If the movement of a transmembrane helix exceeds this limit, associated phospholipids change the partners for fixation or change the orientation of the entire protein molecule.