Impact of Time, Temperature, and Conformational Changes on the Characterization of Cardiotonic Steroid–Na+/K+-ATPase Interactions

Figures

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  Fig. 1. Simulated data of a slower, faster, and very fast kinetics molecule (classification relative to each kobs = kon × Kd + koff) with the equilibrium constant (1.6 × 10− 6 M). In A, B, and C, concentration-response progression curve data fitting with three different molecules showing different values of kon and koff. In D, E, and F, concentration-response curves of different times of respective molecules; G, H, and I are the exponential decay of the apparent Hill coefficient over time of respective molecules. The data were generated with the rate values presented in Table 1, using Eq. 2, with a concentration range from 10− 7 to 10− 3 M, and Bmax was fixed at 100. The concentration-response curve was fitted with Eq. 1, with Bmax fixed at 100. A high concentration was included to ensure accurate determination of the maximal inhibition of each concentration

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  Fig. 2. Simulated data for three molecules (Mol 1, Mol 2, and Mol 3) exhibiting the same affinity at 37 °C. The graph shows the relationship between the equilibrium dissociation constant (Kd, in molar units) and temperature in degrees Celsius. These data were generated using the linear Van’t Hoff equation (Eq. 5), based on the ΔH and ΔS values reported in Table 1

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  Fig. 3. Van’t Hoff plot for three molecules (Mol 1, Mol 2, and Mol 3) with the same affinity at 37 °C. The graph shows the relationship between the natural logarithm of the equilibrium association constant and the inverse of temperature (in Kelvin). These data were generated using the linear Van’t Hoff equation, based on the values reported in Table 1

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  Fig. 4. Free energy of binding for three different molecules (Mol 1, Mol 2, and Mol 3). The graph shows the binding free energy as a function of temperature (in degrees Celsius) for three molecules with distinct energetic profiles. These data were generated using the Gibbs free energy equation, based on the values reported in Table 1

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  Fig. 5. Rate constants (power of 10) at different temperatures for three molecules (Mol 1, Mol 2, and Mol 3). In (A), the association rate constant is shown as a function of temperature (in degrees Celsius). In (B), the dissociation rate constant is presented across different temperatures. These data were generated using the Eyring equation, based on the values reported in Table 1

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  Fig. 6. The image illustrates the free energy that governs the coordination of the drug with a surface receptor. The rate of interaction between the drug (green) and the receptor (blue) will be determined by the molecule with enough energy necessary to overcome the transition state, which can be, for example, the desolvation of a water molecule in the main residue of the binding site, to finally reach the ground state, which forms the ground state interaction. A faster molecule is the one that finds a coordinate with a low energy necessary for the transition state (can be enthalpically or entropically dominated), that can culminate in the same equilibrium affinity/potency, but different rate. At the end, the difference in the energy of association and dissociation results in the energy of the equilibrium ground state

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  Fig. 7. Eyring plot. In (A), the natural logarithm of the association rate constant over by temperature is plotted against the inverse of temperature for three molecules (Mol 1, Mol 2, and Mol 3) with the same affinity at 37 °C. In (B), the natural logarithm of the dissociation rate constant normalized by temperature (ln(k/T)) is shown as a function of the inverse temperature for the three molecules

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  Fig. 8. Transition-state energy barriers of the rate constants. In (A), the Gibbs free energy of activation for the association process is shown as a function of temperature (in degrees Celsius) for three molecules (Mol 1, Mol 2, and Mol 3) with the same affinity at 37 °C. In (B), the Gibbs free energy of activation for the dissociation process is presented as a function of temperature for the three molecules

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  Fig. 9. – Structure of naturally obtained cardiotonic steroids and marked in red all common modifications from the preserved steroid nucleus. In the center, the steroidal nucleus and the numbering of its atoms. Below the nucleus is the representation of its three-dimensional configuration. On the left are the possible substitutions at carbon C3, usually a glycosidic moiety, ranging from a single rhamnose (e.g., ouabain) to three digitoxoses (e.g., digitoxin) naturally occurring, as well as the absence of a glycosidic portion characterized by the presence of a hydroxyl group, leading to the suffix -genin in this cardiotonic steroid (for example, ouabagenin or digitoxigenin). On the right are the substitutions at carbon C17 that define the class: a five-membered ring in the case of cardenolides or a six-membered ring in the case of bufadienolides. The flexibility of some CTS is marked with the mesh sphere and expressed as the number of theoretical rotamers (Nrot), considering 3 theoretical states

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  Fig. 10. Crystallographic structure of the Na+/K+-ATPase in the E2P conformation (PDB ID: 7WZ0 from Squalus acanthias) bound to ouabain is shown. In A, the illustration highlights the α-subunit, composed of the transmembrane helix αTH1–2 (orange), αTH3–4 (light blue), αTH5–6 (purple), and αTH7–8 (brown), as well as the P domain (phosphorylation domain, dark blue), the A domain (actuator domain, yellow), and the N domain (nucleotide-binding domain, red). The β-subunit is depicted in green, and the FXYD subunit in light pink. In B and C, view of the extracellular cavity from the β-subunit as a cartoon and solvent accessible surface, respectively. In D, view of the binding site of ouabain from αTH2 (transparency) with the close (3.5 A) H-bound distance (yellow dashes). In E, only the residues surrounding ouabain (αTH2 was hiding)

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  Fig. 11. Conformational transition from E2P to E2–2 K+Pi (PDB ID: 7WYW and 2ZXE from Squalus acanthias, respectively). The extracellular cavity, viewed from the β-subunit, is represented by the solvent-accessible surface of the E2P conformation (A) and the E2P–2 K conformation (B). The Post–Albers catalytic cycle of Na+/K+-ATPase is shown in (C) (Post et al. 1972; Taniguchi and Post, 1975; source: adapted from Ogawa et al. 2015, https://creativecommons.org/licenses/by/4.0/). Cartoon representations of the transmembrane helices, viewed from the β-subunit, are shown for the E2P conformation (D), the E2P–2 K conformation (E), and the structural alignment of both states (F). A lateral view of the transmembrane helices is presented for the E2P conformation (G), the E2–2 K+Pi conformation (H), and the alignment of the two structures (I)

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  Fig. 12. Conformational effects on CTS binding. In (A) (view from the β-subunit) and (B) (lateral view), alignment of Na+/K+-ATPase in the E2P conformation without ligand (7WYW from Squalus acanthias), E2P in the presence of ouabain (7WZ0 from S. acanthias), and the E2-2 K+Pi conformation in the presence of ouabain (3A3Y from S. acanthias) is shown. In (C) and (D), residues surrounding ouabain in the E2P and E2-2 K+Pi conformations, respectively, are presented. In (E) (view from the β-subunit) and (F) (lateral view), alignment of NKA in the E2P conformation (7WYW from S. acanthias) without ligand, E2P in the presence of bufalin (7DDL from Sus scrofa), and the E2P-2 K conformation in the presence of bufalin (4RES from S. scrofa) is shown. In (G) and (H), residues surrounding bufalin in the E2P and E2P-2 K conformations, respectively, are depicted. Colored arrows indicate the movement associated with the conformational changes of the respective transmembrane helices