Ayami Hirata

Crystal structures of the calcium pump and sarcolipin in the Mg2+-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 Ca2+-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca2+ 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 Ca2+-binding sites ready to accept new cytosolic Ca2+. In the absence of Ca2+ and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca2+), and if millimolar Mg2+ is present, one Mg2+ is expected to occupy one of the Ca2+-binding sites with a millimolar dissociation constant. This Mg2+ accelerates the reaction cycle, not permitting phosphorylation without Ca2+ binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg2+ 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 Ca2+-ATPase, is bound, stabilizing the E1·Mg2+ state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca2+ 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.

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin.

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca2+-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1∼P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg2+, conversion of E2 to E1 pattern occurs (even when Ca2+ is absent) as H+ dissociates from acidic residues. Mutational analysis demonstrates that the Glu309 and Glu771 acidic residues (empty Ca2+-binding sites I and II) are required for stabilization of E2. Glu309 ionization is most important to yield E1. However, a further transition produced by Ca2+ binding to E1 (i.e. E1·2Ca2+) is still needed for catalytic activation. Following ATP utilization, H+/Ca2+ exchange is involved in the transition from the E1∼P·2Ca2+ to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1∼P·2Ca2+ to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca2+. The E2 state and luminal path closure follow removal of conformational constraint by phosphate.

Intermediate Phosphorylation Reactions in the Mechanism of ATP Utilization by the Copper ATPase (CopA) of Thermotoga maritima

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78–2.73 μmol/mg/min in the presence of Cu+ (pH 6, 60 °C) and 0.03–0.08 μmol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [γ-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu·E1 ∼ P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (ΔNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [γ-32P]ATP by the ΔNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the ΔNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in ΔNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an “active” conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu·E1 ∼ P, followed by the E1 ∼ P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained.

Sequential substitution of K(+) bound to Na(+),K(+)-ATPase visualized by X-ray crystallography.

Na+,K+-ATPase transfers three Na+ from the cytoplasm into the extracellular medium and two K+ in the opposite direction per ATP hydrolysed. The binding and release of Na+ and K+ are all thought to occur sequentially. Here we demonstrate by X-ray crystallography of the ATPase in E2·MgF42−·2K+, a state analogous to E2·Pi·2K+, combined with isotopic measurements, that the substitution of the two K+ with congeners in the extracellular medium indeed occurs at different rates, substantially faster at site II. An analysis of thermal movements of protein atoms in the crystal shows that the M3–M4E helix pair opens and closes the ion pathway leading to the extracellular medium, allowing K+ at site II to be substituted first. Taken together, these results indicate that site I K+ is the first cation to bind to the empty cation-binding sites after releasing three Na+.

Visualization of lipid bilayer in the crystals by solvent contrast modulation

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.