Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Pedersen, Per Amstrup; Jørgensen, Jan Stener; Jørgensen, Peter Leth

doi:10.1074/jbc.m005610200

Cited by 77 publications

(85 citation statements)

References 29 publications

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“…The 438 GEAT 441 segment is a part of the nucleotide-binding region of Ca 2ϩ -ATPase and was shown to be in direct proximity to bound ATP (20). Furthermore, even in the E2 state His-1069 is only 18Å away from the Asp residues of the GDGVND motif that are known to be close to the ␤␥-phosphates of the ATP-magnesium complex during catalysis (21). DISCUSSION WNDP has a central role in human copper homeostasis; however, the molecular mechanism of the WNDP-mediated copper transport remains poorly understood.…”

Section: His-1069 Is Not Essential For Phosphorylation Of Wndp By Inomentioning

confidence: 99%

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

Tsivkovskii

Efremov

Lutsenko

2003

Journal of Biological Chemistry

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Section: His-1069 Is Not Essential For Phosphorylation Of Wndp By Inomentioning

confidence: 99%

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

Tsivkovskii

Efremov

Lutsenko

2003

Journal of Biological Chemistry

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“…Asp-351). Analogous mutational analysis of the Na ϩ K ϩ -ATPase (14) indicates that electrostatic interactions around the phosphorylation site may play an important role in substrate positioning and utilization. We considered that their mutation may alter direct interactions with the nucleotide terminal phosphate or ligation of Mg 2ϩ in conjunction with oxygen atoms of the ATP-terminal phosphate.…”

Section: The Ca 2ϩ Atpase Atp Sitementioning

confidence: 99%

Substrate-induced Conformational Fit and Headpiece Closure in the Ca2+ATPase (SERCA)

Ma¹,

Inesi²,

Toyoshima

2003

Journal of Biological Chemistry

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Protection of the Ca2؉ ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca 2؉ , Mg 2؉ , and nucleotide and interpreted as a substrate-dependent conformational change (1). The protected digestion site is located on the loop connecting the A domain and the M3 transmembrane helix. We studied by mutational analysis the protective effect of AMP-PCP, an ATP analog that is not utilized for enzyme phosphorylation. We found that the nucleotide protective effect is interfered with by single mutations of Arg-560 and Glu-439 in the N domain and Lys-352, Lys-684, Thr-353, Asp-703, and Asp-707 in the P domain. This is consistent with a transition from the open to the compact configuration of the ATPase headpiece and approximation of the N and P domains by interactions with the nucleotide adenosine and phosphate moieties, respectively. The A domain-M3 loop is consequently involved. Protection by nucleotide substrate increased following the mutations of Asp-351 (the residue undergoing phosphorylation by ATP) and neighboring Asn-706 to Ala, underlying the importance of side chain specificity in positioning the nucleotide terminal phosphate and limiting the stability of the substrate-enzyme complex. Protection is not observed when AMP-PCP is added in the absence of Ca 2؉ or following mutations (E771Q or N796A) that interfere with Ca 2؉ binding. Therefore, nucleotide binds to the Ca 2؉ -activated enzyme in the open headpiece conformation and the consequent approximation of the N and P domains occurs while the transmembrane domain is still in the Ca 2؉ -bound conformation. Mg 2؉ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions. The sarcoplasmic reticulum (SR)1 ATPase is a 110-kDa enzyme that utilizes 1 mol of ATP for active transport of two calcium ions in exchange for 2 mol of protons. The catalytic cycle begins with cooperative binding of two calcium ions from the cytosolic medium followed by ATP utilization and formation of a phosphorylated enzyme intermediate. The bound calcium is then released onto the luminal side of the SR, and the cycle is completed by hydrolytic cleavage of acylphosphate.The ATPase molecular structure includes a transmembrane region made of 10 clustered helical segments including the Ca 2ϩ binding domain and a cytosolic headpiece including the nucleotide binding (N), phosphorylation (P), and actuator (A) domains. Structural studies have shown that if crystallization is performed in the presence of Ca 2ϩ the three headpiece domains are distinctly separate in an open configuration (2). On the other hand, in the absence of Ca 2ϩ , those domains gather to form a compact headpiece (3, 4). This finding suggests that in solution the three domains undergo fluctuations, yielding different enzyme conformations depending on the presence of specific ligands. Taking advantage of a selective ATPase cleavage (5), it was then shown that the addition of nucleotides in the presence of Mg 2ϩ and Ca 2ϩ ...

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“…Thus, it is highly desirable to investigate the predicted roles of specific residues by mutational analysis. As one relevant example, mutations of residues in the conserved 708 TGDGVNDS sequence in the P domain have already shown that Asp 710 is a Mg 2ϩ binding residue (21). We describe here expression of Na ϩ ,K ϩ -ATPase in the methylotrophic yeast Pichia pastoris and analysis of the wild type protein and the D369N mutant by Fe 2ϩ -catalyzed oxidative cleavages.…”

mentioning

confidence: 99%

“…Thus, expression of functional Na ϩ ,K ϩ -ATPase in P. pastoris at significant levels appeared to be an attractive possibility. Na ϩ ,K ϩ -ATPase has been expressed in many cell types, and used extensively for structure-function analysis, but of most relevance to the present work wild type and mutant Na ϩ ,K ϩ -ATPase proteins have been expressed in Saccharomyces cerevisae and interactions of the pump ligands, ATP, Na ϩ , K ϩ , Mg 2ϩ , and ouabain characterized in detail, particularly by direct binding assays (21,(25)(26)(27)(28)(29). On the other hand, analysis of structural organization and conformational changes by proteolytic cleavage or metalcatalyzed oxidative cleavage has not been described.…”

mentioning

confidence: 99%

Expression of Na+,K+-ATPase in Pichia pastoris

Strugatsky

Gottschalk²,

Goldshleger

et al. 2003

Journal of Biological Chemistry

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Na؉ ,K ؉ -ATPase (pig ␣1,␤1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300 -500 mg of membrane protein with ouabain binding capacity of 30 -50 pmol mg ؊1 . The Na ϩ ,K ϩ -ATPase utilizes the free energy of hydrolysis of ATP to actively transport three intracellular Na ϩ ions and two extracellular K ϩ ions in opposite directions across animal cell membranes. The Na ϩ ,K ϩ -ATPase is a member of the P-type family of cation pumps. The kinetic mechanism of Na ϩ ,K ϩ -ATPase, as of other P-type pumps, involves a phosphoenzyme intermediate and is now largely understood (1, 2). As pointed out by Jencks (3), strict cation and substrate specificities of the phosphorylation and dephosphorylation reactions, and tight coupling of the E 1 7 E 2 conformational changes to cation movements are the essential features of all P-type ion pump mechanisms. These central questions of the energy transduction mechanism of P-type pumps can now be posed in structural terms (4) because of availability of molecular structures of the sarcoplasmic reticulum Ca 2ϩ -ATPase for both E 1 2Ca 2ϩ and E 2 conformations (5, 6).The Ca 2ϩ -ATPase molecule consists of head, stalk, and membrane sectors (5). There are 10 transmembrane segments in the membrane domain with two Ca 2ϩ ions ligated approximately in the center of the bilayer and between transmembrane segments M4, M5, M6, and M8 in the E 1 2Ca 2ϩ conformation. The stalk sector consists of the cytoplasmic extensions of the transmembrane helices, particularly S5 and S4. The cytoplasmic sector consists of three domains, nucleotide binding (N), 1 phosphorylating (P), and anchor or actuator domain (A). Comparison of the crystal structure an E 1 2Ca2ϩ and E 2 conformations shows that in the E 1 conformations the N, P, and A domains are separate, whereas in E 2 conformations the domains are gathered together, moving essentially as rigid bodies (5, 6). Movement of the A domain toward P and N domains in the E 1 3 E 2 transition is associated with a bending of S5 that entails complex movements of several transmembrane segments. This changes the ligation of the occluded Ca 2ϩ ions within the transmembrane segments allowing them to dissociate within the sarcoplasmic reticulum. Whereas this general paradigm clearly applies to the other P-type pumps, not all features are explained by the crystal structures. As one example, phosphorylation by ATP requires close proximity of the nucleotide binding N and phosphorylation P domains, but this is not observed in the E 1 2Ca 2ϩ (Protein Data Bank code 1EUL) structure. In addition, of course, there are the specific features of other ion pumps particularly the cation selectivities,

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Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Cited by 77 publications

References 29 publications

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

Substrate-induced Conformational Fit and Headpiece Closure in the Ca2+ATPase (SERCA)

Expression of Na+,K+-ATPase in Pichia pastoris

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