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.
Highlights
ATP synthases, the universal energy conversion machinery in all living cells, synthesize ATP by a rotational motion [1], inducing the catalytic reaction of synthesizing ATP by phosphorylating ADP
This double mutant binds ATP two orders of magnitude stronger (Kd = 52 nM) [27] than the wild type (Kd = 4.3 μM) [22] protein. Structural reasons for these different binding strengths of the ε subunit to ATP are not obvious. To understand these experimental results, we carried out conventional MD simulations for the R103A/R115A mutant of the ε subunit from thermophilic Bacillus PS3
As we have previously shown for the wild type ε subunit from thermophilic Bacillus PS3 [30], we observe a high probability of Mg2+ binding in a second sphere coordination (Fig 2a)
Summary
ATP synthases, the universal energy conversion machinery in all living cells, synthesize ATP by a rotational motion [1], inducing the catalytic reaction of synthesizing ATP by phosphorylating ADP. This rotational motion is induced by an electrochemical proton [2] or sodium ion [3] gradient; the molecular and energetic basis for the ion selectivity have been characterized previously [4]. To prevent the waste of ATP, ATPase activity in bacteria and mammals is supressed by common and distinct regulatory mechanisms. The ATPase activity in bacterial cells is regulated by the ε subunit, sensing the ATP concentration. A large conformational change from the non-inhibitory down- to the inhibitory up-state is induced, if the ATP
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