Microenvironment of the High Affinity ATP-Binding Site of Na+/K+-ATPase Is Slightly Acidic

Linnertz, Holger; Lanz, Edvard; Gregor, Martin; Antolović, Roberto; Krumscheid, Rita; Obšil, Tomáš; Slavík, Jan; Kovarik, Zeljka; Schoner, Wilhelm; Amler, Evžen

doi:10.1006/bbrc.1998.9874

Cited by 5 publications

(2 citation statements)

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“…Binding of the first two Na + is electroneutral (like binding of 2 K + ions or their congeners); binding of the third Na + is electrogenic with a dielectric coefficient of 0.25 [19,95]. Occupation of the third, highly selective Na + site, which is formed by transmembrane parts of the Na,K-ATPase [13,43,81], is strictly correlated with an effect on the fluorescent FITC label that is bound to a lysine, K501, inside the nucleotide binding site [21,47,60]. The mechanism of this label (its chromophore is a fluorescein) is that of a pH indicator which detects (small) changes of the local proton concentration.…”

Section: Functional Properties With Structural Implicationsmentioning

confidence: 99%

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

Apell

Karlish

2001

Journal of Membrane Biology

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Abstract.A full understanding of the molecular mechanism of ion transport and energetics of the Na,K-ATPase will require both structural and functional data. During recent years biophysical methods have provided a number of important pieces of information on ion binding and release and the charge transfer process. This allows the formulation of kinetic models of the transport process. Although a breakthrough has not been obtained due to the lack of detailed knowledge on the threedimensional structure with a resolution high enough to identify the ion-binding sites and the transport pathway, the functional information has structural implications that create constraints on possible mechanisms of active transport. Here we describe briefly contributions of some biophysical methods to our conceptual understanding of the ion transport process.

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Section: Functional Properties With Structural Implicationsmentioning

confidence: 99%

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

Apell

Karlish

2001

Journal of Membrane Biology

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“…Experiments with pyridoxal 5′‐diphospho‐5′‐adenosine and pyridoxal phosphate suggested that Lys 480 recognized the phosphate moiety of the nucleotide 80. The facts that ATP prevents modification of Lys 501 (or Lys 515 ) by FITC81–83 and that fluorescence of FITC attached to this residue cannot be quenched by antifluorescein84 led to the conclusion that Lys 501 is localized in the depth of the ATP‐binding pocket. Chemical crosslink connecting Lys 480 and Lys 501 suggested that the distance between these residues is 1.4 nm 85.…”

Section: Introductionmentioning

confidence: 99%

ATP‐binding to P‐type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

Kubala

2006

Proteins

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P-type ATPases form a large family of cation translocating ATPases. Recent progress in crystallography yielded several high-resolution structures of Ca(2+)-ATPase from sarco(endo)plasmic reticulum (SERCA) in various conformations. They could elucidate the conformational changes of the enzyme, which are necessary for the translocation of cations, or the mechanism that explains how the nucleotide binding is coupled to the cation transport. However, crystals of proteins are usually obtained only under conditions that significantly differ from the physiological ones and with ligands that are incompatible with the enzyme function, and both of these factors can inevitably influence the enzyme structure. Biochemical (such as mutagenesis, cleavage, and labeling) or spectroscopic experiments can yield only limited structural information, but this information could be considered relevant, because measurement can be performed under physiological conditions and with true ligands. However, interpretation of some biochemical or spectroscopic data could be difficult without precise knowledge of the structure. Thus, only a combination of both these approaches can extract the relevant information and identify artifacts. Briefly, there is good agreement between crystallographic and other experimental data concerning the overall shape of the molecule and the movement of cytoplasmic domains. On the contrary, the E1-AMPPCP crystallographic structure is, in details, in severe conflict with numerous spectroscopic experiments and probably does not represent the physiological state. Notably, the E1-ADP-AlF(4) structure is almost identical to the E1-AMPPCP, again suggesting that the structure is primarily determined by the crystal-growth conditions. The physiological relevance of the E2 and E2-P structures is also questionable, because the crystals were prepared in the presence of thapsigargin, which is known to be a very efficient inhibitor of SERCA. Thus, probably only crystals of E1-2Ca conformation could reflect some physiological state. Combination of biochemical, spectroscopic, and crystallographic data revealed amino acids that are responsible for the interaction with the nucleotide. High sequence homology of the P-type ATPases in the cytoplasmic domains enables prediction of the ATP-interacting amino acids also for other P-type ATPases.

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ATP binding site on the C-terminus of the vanilloid receptor

Grycová

Lánský

Friedlova

et al. 2007

Archives of Biochemistry and Biophysics

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Microenvironment of the High Affinity ATP-Binding Site of Na+/K+-ATPase Is Slightly Acidic

Cited by 5 publications

References 26 publications

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

ATP‐binding to P‐type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

ATP binding site on the C-terminus of the vanilloid receptor

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