Ca2+-ATPase of muscle sarcoplasmic reticulum is an ATP-powered Ca2+-pump that establishes a >10,000 fold concentration gradient across the membrane. Its crystal structures have been determined for 9 ...different states that cover nearly the entire reaction cycle. Presented here is a brief structural account of the ion pumping process, with emphasis on why the structure has to be so.
Ca2+-ATPase of muscle sarcoplasmic reticulum is an ATP-powered Ca2+-pump that establishes a >10,000-fold concentration gradient across the membrane. Its crystal structures have been determined for ...nine different states that cover nearly the entire reaction cycle. Presented here is a brief structural account of the ion pumping process, which is achieved by a series of very large domain rearrangements.
Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across ...the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.
Sodium-potassium ATPase is an ATP-powered ion pump that establishes concentration gradients for Na+ and K+ ions across the plasma membrane in all animal cells by pumping Na+ from the cytoplasm and K+ ...from the extracellular medium. Such gradients are used in many essential processes, notably for generating action potentials. Na+, K+-ATPase is a member of the P-type ATPases, which include sarcoplasmic reticulum Ca2+-ATPase and gastric H+, K+-ATPase, among others, and is the target of cardiac glycosides. Here we describe a crystal structure of this important ion pump, from shark rectal glands, consisting of - and -subunits and a regulatory FXYD protein, all of which are highly homologous to human ones. The ATPase was fixed in a state analogous to E2·2K+·Pi, in which the ATPase has a high affinity for K+ and still binds Pi, as in the first crystal structure of pig kidney enzyme at 3.5 Å resolution. Clearly visualized now at 2.4 Å resolution are coordination of K+ and associated water molecules in the transmembrane binding sites and a phosphate analogue (MgF42-) in the phosphorylation site. The crystal structure shows that the -subunit has a critical role in K+ binding (although its involvement has previously been suggested) and explains, at least partially, why the homologous Ca2+-ATPase counter-transports H+ rather than K+, despite the coordinating residues being almost identical.
The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, ...generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.
Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+ transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates ...Ca2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.
Na+,K+‐ATPase (NKA) is one of the most important members of the P‐type ion‐translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell ...membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K+ state solved by cryo‐electron microscopy. It is a stable state with two occluded K+ after transferring three Na+ into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K+, linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K+ relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K+ into the cytoplasm, more than 100 times.
Na+,K+‐ATPase establishes concentration gradients of Na+ and K+ across the plasma membrane. Cryo‐EM structures of Na+,K+‐ATPase in the E2·2K+ state and after addition of ATP explain how the release of inorganic phosphate locks the extracellular gate to occlude bound K+, and why the addition of ATP prompts the release of K+ into the cytoplasm.
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.
•Long awaited crystal structure of Ca2+-ATPase in the E1·Mg2+ state has appeared.•Sarcolipin and phospholamban bind to Ca2+-ATPase in the E1·Mg2+ state.•Adenine nucleotides and their TNP derivatives ...bind in surprisingly different ways.
P-type ATPases are ATP-powered ion pumps, classified into five subfamilies (PI–PV). Of these, PII-type ATPases, including Ca2+-ATPase, Na+,K+-ATPase and gastric H+,K+-ATPase, among others, have been the most intensively studied. Best understood structurally and biochemically is Ca2+-ATPase from sarcoplasmic reticulum of fast twitch skeletal muscle (sarco(endo)plasmic reticulum Ca2+-ATPase 1a, SERCA1a). Since publication of the first crystal structure in 2000, it has continuously been a source of excitement, as crystal structures for new reaction intermediates always show large structural changes. Crystal structures now exist for most of the reaction intermediates, almost covering the entire reaction cycle. This year the crystal structure of a missing link, the E1·Mg2+ state, finally appeared, bringing another surprise: bound sarcolipin (SLN). The current status of two other important PII-type ATPases, Na+,K+-ATPase and H+,K+-ATPase, is also briefly described.
The sodium pump (Na
, K
-ATPase, NKA) is vital for animal cells, as it actively maintains Na
and K
electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) ...such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF
complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K
-antagonism and suggest a route to isoform specific CTSs.
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