Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues

P-type ion transporting ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes. Transfer of bound cations to the lumenal or extracellular side occurs while the ATPase is phosphorylated. Here we report at 2.3 A resolution the structure of the calcium-ATPase of skeletal muscle sarcoplasmic reticulum, a representative P-type ATPase that is crystallized in the absence of Ca2+ but in the presence of magnesium fluoride, a stable phosphate analogue. This and other crystal structures determined previously provide atomic models for all four principal states in the reaction cycle. These structures show that the three cytoplasmic domains rearrange to move six out of ten transmembrane helices, thereby changing the affinity of the Ca2+-binding sites and the gating of the ion pathway. Release of ADP triggers the opening of the lumenal gate and release of phosphate its closure, effected mainly through movement of the A-domain, the actuator of transmembrane gates.     

Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence

We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.     

Structural changes in the calcium pump accompanying the dissociation of calcium

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca(2+) ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A resolution in a Ca(2+)-free (E2) state, and compare it with that determined previously for the Ca(2+)-bound (E1Ca(2+)) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca(2+). Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.     

The structural basis of calcium transport by the calcium pump

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.     

Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase

Cation pumps bind and translocate ions with the intermediate formation of a phosphoenzyme. In spite of extensive knowledge of the primary and even secondary structures of several of these cation transport enzymes, however, no high affinity cation binding sites have yet been determined. Here we report the use of oligonucleotide-directed, site-specific mutagenesis to identify the amino acids involved in Ca2+ binding in one of these transport enzymes, the Ca2+-ATPase of sarcoplasmic reticulum. Alteration of Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 or Glu 908, each of which is predicted to lie near the centre of the transmembrane domain in putative transmembrane sequences M4, M5, M6 and M8 resulted in complete loss of Ca2+ transport function and of Ca2+-dependent phosphorylation of the enzyme by ATP. Phosphorylation of each of the mutant enzymes with inorganic phosphate was observed, however, even in the presence of Ca2+, which inhibits phosphorylation in the wild-type enzyme possessing an intact high affinity Ca2+-binding site. These results suggest that at least six polar, oxygen-containing residues lying near the centre of the transmembrane domain provide ligands for one or both of the two high affinity Ca2+ binding sites in the Ca2+-ATPase.     

Occluded bound calcium on the phosphorylated sarcoplasmic transport ATPase

The Ca2+ + Mg2+-activated ATPase of the sarcoplasmic reticulum is responsible for the active Ca2+ transport of this membrane system, the key feature of which is the formation of an energy-rich phosphorylated transport enzyme (EP) and its conversion. To understand the Ca2+-transport mechanism, it is essential to clarify the behaviour of this intermediate in relation to such ligands as ATP, ADP, Mg2+ and, particularly, Ca2+. Recent kinetic studies on the phosphate turnover of this system suggested a relatively slow rate of Ca2+ dissociation from the phosphorylated enzyme, which possibly indicated Ca2+ binding in some occluded form with the intermediate. Here we report direct measurements of the binding and release of Ca2+ during phosphorylation of the sarcoplasmic transport enzyme. The results indicate an occlusion of the Ca2+ binding, accompanying an initial configurational change of the enzyme induced by the energy-rich phosphoryl transfer.     

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.     

Crystal structure of the plasma membrane proton pump

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Crystal structure of the MgtE Mg2+ transporter

The magnesium ion Mg2+ is a vital element involved in numerous physiological processes. Mg2+ has the largest hydrated radius among all cations, whereas its ionic radius is the smallest. It remains obscure how Mg2+ transporters selectively recognize and dehydrate the large, fully hydrated Mg2+ cation for transport. Recently the crystal structures of the CorA Mg2+ transporter were reported. The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains, and human homologues have been functionally characterized and suggested to be involved in magnesium homeostasis. However, the MgtE transporters have not been thoroughly characterized. Here we determine the crystal structures of the full-length Thermus thermophilus MgtE at 3.5 A resolution, and of the cytosolic domain in the presence and absence of Mg2+ at 2.3 A and 3.9 A resolutions, respectively. The transporter adopts a homodimeric architecture, consisting of the carboxy-terminal five transmembrane domains and the amino-terminal cytosolic domains, which are composed of the superhelical N domain and tandemly repeated cystathionine-beta-synthase domains. A solvent-accessible pore nearly traverses the transmembrane domains, with one potential Mg2+ bound to the conserved Asp 432 within the pore. The transmembrane (TM)5 helices from both subunits close the pore through interactions with the 'connecting helices', which connect the cystathionine-beta-synthase and transmembrane domains. Four putative Mg2+ ions are bound at the interface between the connecting helices and the other domains, and this may lock the closed conformation of the pore. A structural comparison of the two states of the cytosolic domains showed the Mg2+-dependent movement of the connecting helices, which might reorganize the transmembrane helices to open the pore. These findings suggest a homeostasis mechanism, in which Mg2+ bound between cytosolic domains regulates Mg2+ flux by sensing the intracellular Mg2+ concentration. Whether this presumed regulation controls gating of an ion channel or opening of a secondary active transporter remains to be determined.     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.     

Crystal structure of the sodium-potassium pump

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.     

Crystal structure of a copper-transporting PIB-type ATPase

Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 ? resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes' and Wilson's diseases.     

Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate

D-myo-inositol-1,4,5-trisphosphate (InsP3) is a putative intracellular second messenger for the mobilization of Ca2+ from intracellular stores, in particular, the endoplasmic reticulum. Specific binding sites on the endoplasmic reticulum may participate in the InsP3-induced release of Ca2+ from the Ca2+ pool. To examine the specific binding sites on the endoplasmic reticulum, we synthesized an arylazide derivative of InsP3 for photoaffinity labelling; InsP3 coupled to p-azidobenzoic acid (InsP3-pAB) using N,N'-carbonyldiimidazole (CDI) was obtained at a 9-11% yield. Here, we report that InsP3-pAB, but not an arylazide derivative of inositol-1,4-bisphophate (Ins(1,4)P2), causes the irreversible inhibition of InsP3-induced release of Ca2+ in saponin-permeabilized photo-irradiated macrophages. The irreversible inhibition by InsP3-pAB after photo-irradiation was prevented by a 10-fold excess of unmodified InsP3.     

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.     

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5?? resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.     

Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.     

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.     

Interaction of Divalent Metal Ions with the Adenosine Triphosphate Measured Using Nuclear Magnetic Resonance

Theinteractionofadenosinetriphosphatewithdivalentmetalionsisimportantinbiochemicalfunctions.TheintracellularfreeMg2 canbeestimated[1]byseparatingtheα-andβ-phosphatepeaksoftheATPNMR31PspectrumandbyknowingtheMgATPdissociationconstant.TheneedfordivalentmetalionsforallenzymaticreactionsinvolvingATPiswellknown,buttheroleofthemetalionsisnotclear[2].ThisstudywasconductedtoobtaininformationconcerningchangesinthestructureofadenosinetriphosphatecausedbyinteractionwithdivalentmetalionsMg2 ,Ca2 ,…     

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.