Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na+/K+-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

• Keywords
• C45 loop, Na+/K+-ATPase, binding site, cisplatin, cysteine mutants, sodium pump,
• MeSH
• Cisplatin chemistry   pharmacology   MeSH
• Cysteine antagonists & inhibitors   metabolism   MeSH
• Cytoplasm drug effects   metabolism   MeSH
• Mass Spectrometry MeSH
• Mutagenesis, Site-Directed MeSH
• Mice MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Molecular Dynamics Simulation MeSH
• Sodium-Potassium-Exchanging ATPase antagonists & inhibitors   genetics   metabolism   MeSH
• Binding Sites drug effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cisplatin MeSH
• Cysteine MeSH
• Antineoplastic Agents MeSH
• Sodium-Potassium-Exchanging ATPase MeSH

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg2+•ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg543 and Asp611-triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

• Keywords
• Hinge movement, M4M5 loop, Na+/K+-ATPase phosphorylation, Open and closed conformations,
• Publication type
• Journal Article MeSH

We examined the inhibitory effects of three flavonolignans and their dehydro- derivatives, taxifolin and quercetin on the activity of the Na(+)/K(+)-ATPase (NKA). The flavonolignans silychristin, dehydrosilychristin and dehydrosilydianin inhibited NKA with IC50 of 110 ± 40 μM, 38 ± 8 μM, and 36 ± 14 μM, respectively. Using the methods of molecular modeling, we identified several possible binding sites for these species on NKA and proposed the possible mechanisms of inhibition. The binding to the extracellular- or cytoplasmic C-terminal sites can block the transport of cations through the plasma membrane, while the binding on the interface of cytoplasmic domains can inhibit the enzyme allosterically. Fluorescence spectroscopy experiments confirmed the interaction of these three species with the large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45). The flavonolignans are distinct from the cardiac glycosides that are currently used in NKA treatment. Because their binding sites are different, the mechanism of inhibition is different as well as the range of active concentrations, one can expect that these new NKA inhibitors would exhibit also a different biomedical actions than cardiac glycosides.

• Keywords
• Na+/K+-ATPase, binding sites, flavonolignans, inhibition, sodium pump,
• Publication type
• Journal Article MeSH

A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

• MeSH
• Adenosine Triphosphate metabolism   pharmacology   MeSH
• Acrylamide metabolism   pharmacology   MeSH
• Cytoplasm metabolism   MeSH
• Fluorescence MeSH
• Magnesium metabolism   pharmacology   MeSH
• Iodides metabolism   pharmacology   MeSH
• Protein Conformation drug effects   MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Mice MeSH
• Surface Properties MeSH
• Sodium-Potassium-Exchanging ATPase chemistry   genetics   metabolism   MeSH
• Static Electricity * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Acrylamide MeSH
• Magnesium MeSH
• Iodides MeSH
• Ligands MeSH
• Sodium-Potassium-Exchanging ATPase MeSH