The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase

Abstract

The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

Fig 1. Input structures for the simulations.

In a) the whole protein-ATP complex is shown. In b-d) the initial binding site structures for the freely distributed Mg²⁺ case, the MgATP:Oα/Oβ and MgATP:Oβ/Oγ coordination are shown, respectively. Water molecules coordinating the Mg²⁺ ion and residues coordinating ATP are shown in licorice, while the Mg²⁺ ion is shown in VdW spheres.

Fig 2. Distance distribution of the Mg²⁺ ion and free energy results.

Distance distribution when the Mg²⁺ ion a) is bound in a second sphere coordination to ATP, b) is bound in a first sphere coordination to ATP:Oα/Oβ and c) is bound in a first sphere coordination to ATP:Oβ/Oγ; ATP is bound to the protein in all cases. In a)–c) the black, red, green, blue and orange lines represent the minimal distance distributions of a Mg²⁺ ion towards ATP:O2’, ATP:O3’, ATP:Oα, ATP:Oβ and ATP:Oγ, respectively. The before mentioned data were extracted from conventional MD simulations. In d) the free energy calculations (Thermodynamic Integration) for the binding of Mg²⁺ towards ATP in second sphere coordination, when bound to ATP:Oα/Oβ or ATP:Oβ/Oγ are shown, respectively. It should be noted that in Fig b) the blue and the green lines are overlapping.

Fig 3. Distance distribution of protein-ATP interactions.

Distance distribution of protein-ATP interactions of the Mg²⁺ bound to ATP:Oα/Oβ. Dotted lines represent distances found in the crystal structure of the wild type protein. The histogram in the top left represents nucleoside–protein interaction (black: ATP:N6 –D89:O, red: ATP:O2’–E:83:Oεx, green: ATP:O3’–E83:Oεx, blue: D89:N—ATP:N1, violet: R92:NHx—ATPO4’, cyan: R92:NHx—ATP:N3/7/9 and orange: R126:NHx—ATP:O5’). The three other diagrams represent protein—ATP:Oα/β/γ interactions (black: R92:Nε, red: R92:NHx, green: R99:Nε, blue: R99:NHx, brown: R122:Nε, cyan: R122:NHx, magenta: R126:Nε and orange: R126:NHx), respectively. The corresponding figures for the Mg²⁺ freely distributed state and Mg²⁺ coordinated to ATP:Oβ/Oγ are shown in S2 and S4 Figs in the Supporting Information, respectively. The corresponding data for the single runs is shown in S5 (Mg²⁺ not bound in first sphere), S6 (Mg²⁺ bound to ATP:Oα/Oβ) and S7 Figs (Mg²⁺ bound to ATP:Oβ/Oγ), respectively.

Fig 4. Comparison of wild type and mutant ATP binding site.

ATP binding site of a representative snapshot of a) the wild type ε subunit [30] and b) the R103A/R115A double mutant derived by MD simulations. A structural comparison of the crystal structure and the R103A/R115A mutant is shown in the supporting information (S8 Fig). Figures containing molecular information have been produced using VMD [58].