Natalya U. Fedosova

Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

The Na+,K+-ATPase maintains electrochemical gradients for Na+ and K+ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na+,K+-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na+,K+-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2–MgFx–ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg2+. K+ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K+–Mg2+ competition and explain the well-known antagonistic effect of K+ on CTS binding.

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase.

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.

Crystal Structure of Na+, K+-ATPase in the Na+-Bound State

Pumping Out Sodium Mammalian cells contain relatively high concentrations of potassium but low concentrations of sodium. This balance is maintained by an ion pump, the Na+, K+–adenosine triphosphatase, in an adenosine triphosphate–driven transport cycle that results in the export of three sodium ions and the import of two potassium ions. Structures of potassium-bound conformations of the pump have been determined. Now, Nyblom et al. (p. 123, published online 19 September) report on the high-resolution crystal structure of a Na+-bound conformation, which reveals conformational changes associated with Na+ binding. The location of three bound sodium ions and the mechanism of sodium release in a key plasma membrane ion pump are revealed. The Na+, K+–adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na+ and K+ across the plasma membrane—a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K+-bound or ouabain-blocked forms. We present the crystal structure of a Na+-bound Na+, K+-ATPase as determined at 4.3 Å resolution. Compared with the K+-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na+ sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na+ from IIIb through IIIa, followed by exchange of Na+ for K+ at sites I and II, is suggested.

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

Significance Na+,K+-ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na+ and K+ across the membrane of animal cells. Cardiotonic steroids constitute a broad class of specific Na+,K+-ATPase inhibitors, including drugs of clinical importance with multiple physiological effects. The existence of several endogenous cardiotonic steroids suggests their involvement in health and disease mediated by various signaling pathways, but the structure–activity relationships are not yet understood. Using X-ray crystallography and analysis of binding kinetics, we characterize Na+,K+-ATPase complexes with a total of five cardiotonic steroids, showing variations in glycosylation, steroid core substituents, and structure of the lactone substituent. This insight is highly relevant for the understanding of physiological effects and future drug development based on cardiotonic steroids. Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na+,K+-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na+,K+-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P–ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg2+ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K+ in the E2P–bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1–2.

Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin

Changes in the absorbance spectrum of aquo-cobalamin (Cbl·OH2) revealed that its binding to transcobalamin (TC) is followed by slow conformational reorganization of the protein-ligand complex (Fedosov, S. N., Fedosova, N. U., Nexø, E., and Petersen, T. E. (2000)J. Biol. Chem. 275, 11791–11798). Two phases were also observed for TC when interacting with a Cbl-analogue cobinamide (Cbi), but not with other cobalamins. The slow phase had no relation to the ligand recognition, since both Cbl and Cbi bound rapidly and in one step to intrinsic factor (IF) and haptocorrin (HC), namely the proteins with different Cbl specificity. Spectral transformations observed for TC in the slow phase were similar to those upon histidine complexation with Cbl·OH2 and Cbi. In contrast to a closed structure of TC·Cbl·OH2, the analogous IF and HC complexes revealed accessibility of Cbls upper face to the external reagents. The binders decreased sensitivity of adenosyl-Cbl (Cbl·Ado) to light in the range: free ligand, IF·, HC·, TC·Cbl·Ado. The spectrum of TC·Cbl·Ado differed from those of IF and HC and mimicked Cbl·Ado participating in catalysis. The above data suggest presence of a histidine-containing cap shielding the Cbl-binding site in TC. The cap coordinates to certain corrinoids and, possibly, produces an incapsulated Ado-radical when Cbl·Ado is bound.

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio.

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics: A study employing recombinant human transcobalamin and intrinsic factor

Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

Synthesis and evaluation of cardiac glycoside mimics as potential anticancer drugs

The cardiac glycoside digitoxin, consisting of a steroid core linked to a labile trisaccharide, has been used for centuries for the treatment of congestive heart failure. The well known pharmacological effect is a result of the ability of cardiac glycosides to inhibit the Na(+), K(+)-ATPase. Within recent years cardiac glycosides have furthermore been suggested to possess valuable anticancer activity. To mimic the labile trisaccharide of digitoxin with a stabile carbohydrate surrogate, we have used sulfur linked ethylene glycol moieties of varying length (mono-, di-, tri- or tetra-ethylene glycol), and furthermore used these linkers as handles for the synthesis of bivalent steroids. The prepared compounds were evaluated for their potencies to inhibit the Na(+), K(+)-ATPase and for their cytotoxic effect on cancerous MCF-7 cells. A clear trend is observed in both inhibition and cytotoxic effect, where the bioactivity decreases as the size increases. The most potent Na(+), K(+)-ATPase inhibitors are the compounds with the shortest ethylene glycol chain (K(app) 0.48 μM) and thiodigitoxigenin (K(app) 0.42 μM), which both are comparable with digitoxigenin (K(app) 0.52 μM). For the cancer cell viability assay the shortest mimics were found to have highest efficacy, with the best ligand having a monoethylene glycol unit (IC(50) 0.24 μM), which was slightly better than digitoxigenin (IC(50) 0.64 μM), while none of the novel cardiac glycoside mimics display an in vitro effect as high as digitoxin (IC(50) 0.02 μM).

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.

A blue corrinoid from partial degradation of vitamin B12 in aqueous bicarbonate: spectra, structure, and interaction with proteins of B12 transport.

Cobalamin (Cbl) is a complex cofactor produced only by bacteria but used by all animals and humans. Cyanocobalamin (vitamin B(12), CNCbl) is one commonly isolated form of cobalamin. B(12) belongs to a large group of corrinoids, which are characterized by a distinct red color conferred by the system of conjugated double bonds of the corrin ring retaining a Co(III) ion. A unique blue Cbl derivative was produced by hydrolysis of CNCbl in a weakly alkaline aqueous solution of bicarbonate. This corrinoid was purified and isolated as dark blue crystals. Its spectroscopic analysis and X-ray crystallography revealed B-ring opening with formation of 7,8-seco-cyanocobalamin (7,8-sCNCbl). The unprecedented structural change was caused by cleavage of the peripheral C-C bond between saturated carbons 7 and 8 of the corrin macrocycle accompanied by formation of a C═C bond at C7 and a carbonyl group at C8. Additionally, the C-amide was hydrolyzed to a carboxylic acid. The extended conjugation of the π-system caused a considerable red shift of the absorbance spectrum. Formation and degradation of 7,8-sCNCbl were analyzed qualitatively. Its interaction with the proteins of mammalian Cbl transport revealed both a slow binding kinetics and a low overall affinity. The binding data were compared to those of other monocarboxylic derivatives and agreed with the earlier proposed scheme for two-step ligand recognition. The obtained results are consistent with the structural models of 7,8-sCNCbl and the transport proteins intrinsic factor and transcobalamin. Potential applications of the novel derivative for drug conjugation are discussed.