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

Toyoshima, Chikashi; Nakasako, Masayoshi; Nomura, Hiromi; Ogawa, Hirotaka

doi:10.1038/35015017

Cited by 1,759 publications

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“…As it was unambiguously demonstrated by previous binding measurements [7] and crystallographic analysis [11] that SERCA binds two Ca 2+ per mole, we refer to this value as 100% saturation, and show in Fig. 2 …”

Section: Binding and Atpase Measurementsmentioning

confidence: 67%

“…(10) (11) where N S is the total concentration of SERCA molecules. Since the measured Ca 2+ binding isotherms for SERCA are normalized with respect to the maximum saturating values, for comparison the calculated results also need to be expressed as normalized Ca 2+ binding curves.…”

Section: Proposed Statistical Mechanics Analysismentioning

confidence: 99%

“…Binding is pH dependent [8,9] and occurs with high affinity and positive cooperativity. The two Ca 2+ binding sites reside within the membrane bound region of the ATPase [10][11][12]. Ca 2+ complexation at the first site is achieved with oxygen atoms derived from Asn768, Glu771, Thr799, Asp800, Glu908 and two water molecules.…”

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confidence: 99%

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Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Zafar¹,

Hussain²,

Liu³

et al. 2008

Archives of Biochemistry and Biophysics

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Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca 2+ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca 2+ binding isotherms obtained by equilibrium measurements in the presence of various H + and Mg 2+ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca 2+ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation. KeywordsCa 2+ ATPase; Ca 2+ binding; H + /Ca 2+ exchange; Statistical analysis Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. The characteristics of this binding are of utmost importance, as they determine catalytic activation, specificity and stoichiometry of transport and countertransport, and efficiency of ATP utilization. They also correspond to structural features of the enzyme protein with regard to binding affinity and specificity, as well as conformational consequences of binding resulting in catalytic activation. We provide here a detailed analysis of Ca 2+ binding to the Ca 2+ transport ATPase of sarco-endoplasmic reticulum (SERCA) [1,2]. The SERCA isoform of skeletal muscle is a well characterized enzyme [3,4] that utilizes the free energy of ATP for Ca 2+ transport against a concentration gradient. The functional unit is a protein monomer comprising 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P" and "A") protruding from the cytosolic surface of the membrane [5,6]. The ATPase cycle begins with high affinity binding of Ca 2+ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate. Isomerization of the phosphoenzyme intermediate is then coupled to active transport of the bound Ca 2+ across the membrane ("inside"). Hydrolytic cleavage of the phosphoenzyme is the final step that allows enzyme turnover. The high Ca 2+ affinity state of the enzyme is generally referred to as E 1 , and the low affinity as E 2 . Methods Ca 2+ binding and ATPase measurementsVesicular fragments of sarcoplasmic reticulum membrane were obtained from rabbit hind leg muscle as previously described [19]. SERCA accounts for 50-60% of total protein in this preparation. Total protein was determined by the Folin method, standardized with serum albumin.Calcium binding was measured under equilibrium conditions by molecular sieve chromatography [7,20] We now discuss the energies associated with the proposed SERCA species. The energy of an unoccupied species (E) is set as the reference and the energies of all other species are measured with respect to it. ...

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Section: Binding and Atpase Measurementsmentioning

confidence: 67%

Section: Proposed Statistical Mechanics Analysismentioning

confidence: 99%

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Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Zafar¹,

Hussain²,

Liu³

et al. 2008

Archives of Biochemistry and Biophysics

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“…In the binding sequence of Na + this site would be related to the second ion bound. The first Na + ion is assumed to bind to a site formed by transmembrane ␣-helices (which could be M4-M6 and M8 according to [37]). In the case of Na,K-ATPase this site is not accessible for di-or trivalent cations.…”

Section: Discussionmentioning

confidence: 99%

“…Menguy and collaborators suggested that the cytoplasmic loop located between transmembrane segments 6 and 7, L6/7, controls the access to the first and second Ca 2+ binding site, and that this process could be related with the occlusion of the first Ca 2+ by a movement of the L6/7 over the first site, thus opening the second [24]. The recent publication of the crystal structure of the SR Ca-ATPase has proven the side by side binding of Ca 2+ ions, although the loop L6/7 has been given a different assignment as to how it affects Ca 2+ binding [37]. Due to the To determine the half-saturating Na + concentrations, K Na , the experiments were fitted with Hill functions (Eq.…”

Section: Binding Of Na + In the Absence Of Other Cationsmentioning

confidence: 99%

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Schneeberger

Apell

2001

Journal of Membrane Biology

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Abstract.In the E 1 state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di-and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br 2 -Titu 3+ is bound with the highest affinity (< M), among the tested divalent cations Ca 2+ binds the strongest, and Na + binds with about the same equilibrium dissociation constant as Mg 2+ (∼0.8 mM). For alkali ions it exhibits binding affinities following the order of Rb + Ӎ K + > Na + > Cs + > Li + . The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na + ions the equilibrium dissociation constant is < 0.01 mM. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na + , no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mM.

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Calcium Pump ( ATPase ) of Sarcoplasmic Reticulum

Toyoshima¹

2004

Handbook of Metalloproteins

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Ca 2+ ‐ATPase of sarcoplasmic reticulum is a membrane protein of 110 kDa and pumps Ca 2+ from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle, to relax muscle cells. Two Ca 2+ ions can be transported per ATP hydrolyzed and two or three protons are countertransported. The Ca 2+ ‐binding sites are located side by side in the transmembrane region. The crystal structures of this ATPase obtained for Ca 2+ ‐bound and unbound states show that large conformation changes take place during Ca 2+ binding and dissociation.

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

Cited by 1,759 publications

References 47 publications

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Calcium Pump ( ATPase ) of Sarcoplasmic Reticulum

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