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.
Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca 2+ -ATPase, a representative P-type ATPase, in the absence ...of Ca 2+ 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 2+ -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 2+ -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.
Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum is an ATP-driven Ca²⁺ pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca²⁺, the three cytoplasmic ...domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca²⁺-ATPase in the absence of Ca²⁺ stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1-M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1' helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca²⁺-ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations.
Ca2+‐ATPase of sarcoplasmic reticulum is known to pump Mn2+ in addition to Ca2+, but whether its transport mechanism is identical to that of Ca2+ is ambiguous. To clarify, we examined, by atomic ...absorption spectroscopy, competition between Mn2+ and Ca2+ in active transport using vesicles of sarcoplasmic reticulum (SR). Here, we demonstrate that Ca2+‐ATPase transports Ca2+ and Mn2+ concomitantly but has a much lower affinity for Mn2+ (apparent Kd ~ 0.5 mm). Stoichiometries of transported ions per ATP hydrolysed, Vmax values and activation energies are very similar. Altogether, Ca2+‐ATPase appears to use the same mechanism for transporting Mn2+ as that for Ca2+.
Mn2+ competes with Ca2+ in binding to Ca2+‐ATPase but with a much lower affinity.
Mn2+ and Ca2+ are concomitantly and competitively transported by Ca2+‐ATPase.
Ca2+‐ATPase uses the same transport mechanism for Mn2+ as that for Ca2+.
The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or ...specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.
Background: Na,K-ATPase is stabilized by phosphatidylserine/cholesterol and is stimulated by neutral phospholipids.
Results: Three specific lipid-Na,K-ATPase interactions are detectable that either (a) stabilize the protein or (b) stimulate or (c) inhibit Na,K-ATPase activity, with distinct kinetic mechanisms.
Conclusion: There are separate binding sites for phosphatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and sphingomyelin/cholesterol (inhibitory).
Significance: In physiological conditions, specifically bound lipids may regulate Na,K-ATPase activity.
$Ca^{2+}-ATPase$of sarcoplasmic reticulum is an ATP-powered Ca2+pump but also a H+pump in the opposite direction with no demonstrated functional role. Here, we report ...a$2.4-\ring{A}-resolution$crystal structure of the$Ca^{2+}-ATPase$in the absence of Ca2+stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca2+binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by$Ca^{2+}-release$. They suggest that H+countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca2+-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.
The development of cancer is driven not only by genetic mutations but also by epigenetic alterations. Here, we show that TET1-mediated production of 5-hydroxymethylcytosine (5hmC) is required for the ...tumorigenicity of glioblastoma cells. Furthermore, we demonstrate that chromatin target of PRMT1 (CHTOP) binds to 5hmC. We found that CHTOP is associated with an arginine methyltransferase complex, termed the methylosome, and that this promotes the PRMT1-mediated methylation of arginine 3 of histone H4 (H4R3) in genes involved in glioblastomagenesis, including EGFR, AKT3, CDK6, CCND2, and BRAF. Moreover, we found that CHTOP and PRMT1 are essential for the expression of these genes and that CHTOP is required for the tumorigenicity of glioblastoma cells. These results suggest that 5hmC plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex to selective sites on the chromosome, where it methylates H4R3 and activates the transcription of cancer-related genes.
Heavy metal pumps constitute a large subgroup in P‐type ion‐transporting ATPases. One of the outstanding features is that the nucleotide binding N‐domain lacks residues critical for ATP binding in ...other well‐studied P‐type ATPases. Instead, they possess an HP‐motif and a Gly‐rich sequence in the N‐domain, and their mutations impair ATP binding. Here, we describe 1.85 Å resolution crystal structures of the P‐ and N‐domains of CopA, an archaeal Cu+‐transporting ATPase, with bound nucleotides. These crystal structures show that CopA recognises the adenine ring completely differently from other P‐type ATPases. The crystal structure of the His462Gln mutant, in the HP‐motif, a disease‐causing mutation in human Cu+‐ATPases, shows that the Gln side chain mimics the imidazole ring, but only partially, explaining the reduction in ATPase activity. These crystal structures lead us to propose a role of the His and a mechanism for removing Mg2+ from ATP before phosphoryl transfer.
Homology modeling of the α-subunit of Na+K+-ATPase, a representative member of P-type ion transporting ATPases, was carried out to identify the cation (three Na+and two K+) binding sites in the ...transmembrane region, based on the two atomic models of Ca2+-ATPase (Ca2+-bound form for Na+, unbound form for K+). A search for potential cation binding sites throughout the atomic models involved calculation of the valence expected from the disposition of oxygen atoms in the model, including water molecules. This search identified three positions for Na+and two for K+at which high affinity for the respective cation is expected. In the models presented, Na+- and K+-binding sites are formed at different levels with respect to the membrane, by rearrangements of the transmembrane helices. These rearrangements ensure that release of one type of cation coordinates with the binding of the other. Cations of different radii are accommodated by the use of amino acid residues located on different faces of the helices. Our models readily explain many mutational and biochemical results, including different binding stoichiometry and affinities for Na+and K+.
The sarcoplasmic reticulum Ca²⁺-ATPase transports two Ca²⁺ per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity ...and accessibility for Ca²⁺ by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca²⁺-ATPase in the Ca²⁺-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca²⁺, but only Glu771Gln completely looses the Ca²⁺-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca²⁺ was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca²⁺ and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca²⁺ in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca²⁺ and point to a strong relationship between Ca²⁺-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.
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