Experimental and Theoretical Characterization of the High-Affinity Cation-Binding Site of the Purple Membrane

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.

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Characterization of ruthenium red-binding sites of the Ca2+-ATPase from sarcoplasmic reticulum and their interaction with Ca2+-binding sites

Biochemical Journal ◽

10.1042/bj2870767 ◽

1992 ◽

Vol 287 (3) ◽

pp. 767-774 ◽

Cited By ~ 14

Author(s):

S Corbalan-Garcia ◽

J A Teruel ◽

J C Gomez-Fernandez

Keyword(s):

Sarcoplasmic Reticulum ◽

Binding Sites ◽

Ruthenium Red ◽

Cation Binding ◽

Binding Studies ◽

High Affinity ◽

Cation Binding Site ◽

Kinetics Of ◽

Fast Release

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5′-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

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