Figure 3. Cation binding induces eclipsed conformation of the phosphate chain.

(A) Conformations of Mg-ATP complexes with one and two K⁺ ions bound as inferred from MD simulations; left structure, no K⁺ ion bound in the AG site; right structure, a K⁺ ion is bound in the AG site. The α-phosphate is in on the top, β- and γ-phosphates are below; the α-phosphate is shown in green, β-phosphate in blue, γ-phosphate in red. (B) Distribution histograms for dihedral angles between phosphate groups in ATP, calculated from MD simulations of Mg-ATP with one K⁺ cation bound in the BG site (green) and with two cations bound in the AG and BG sites (red). Normalized histograms of dihedral angle distribution (thin lines) were calculated from MD trajectories and fitted with normal distribution function (thick lines). Dashed lines indicate the centroid values of the fits by Gaussian function. All distributions were fitted with one-term Gaussian models, except for the Ψ^β-γ angle in case of Mg-ATP with two cations bound, this distribution was fitted with a two-term Gaussian, parameters for the highest peak are shown. (C) The phosphate chain of GTP, illustrating the dihedral angle Ψ^α-γ. Dihedral angle is an angle between two planes and is defined by four atoms. In this case, the angle Ψ^α-γ is an angle between the plane that contains atoms P^G, P^A and O^1A (green), and the plane that contains atoms P^A, P^G and O^3G (red). In the fully eclipsed conformation, both P-O bonds are coplanar, so that the two planes overlap and the dihedral angle between them is 0°.

Figure 3—figure supplement 1. Coupling between cation binding in the AG site and rotation of γ-phosphate relative to α- and β-phosphates.

Data from MD simulations with restraints on the positions of K⁺ ions (see the text and Supplementary file 1C). The top graph shows free energy calculated from normalized probabilities of ATP conformations and plotted as function of the dihedral angle between γ- and β-phosphates. The bottom plot displays free energy of coupling the binding of the second K⁺ ion with the γ-phosphate rotation, calculated as the difference between the free energy plots shown on the top graph. The lowest energy value was set to zero. These plots show that the presence of second K⁺ ion in the AG site induces a near-eclipsed state of the phosphate chain, by bringing both Ψ^α-β and Ψ^α-γ angles close to 0°, at the expense of Ψ^β-γ, which increases slightly (see Supplementary file 1D). Binding of the second K⁺ ion in the AG site stabilizes this almost eclipsed state by ~27 meV.