Cracking Controls ATP Hydrolysis in the catalytic domain of a P-ATPase

Membrane transporters are essential for cell homeostasis. Among them P-type ATPases, which couple ATP hydrolysis to solute transport, play a key role. Despite extensive research, the fine mechanisms by which this coupling occurs are not completely understood. In this work, we analyzed the effect of substrate, temperature, and urea on the steady state ATPase activity, tryptophan fluorescence and far-UV ellipticity of the catalytic domain of a thermophilic Cu(I) transport ATPase. Through local frustration analysis in combination with AlphaFold2 prediction, we identified a novel conformation, enabling molecular dynamics simulations of an open/close transition. Our results revealed a “cracking” mechanism as a key step in the catalytic cycle. Furthermore, we developed a model that fully describes all the experimental observations. These findings reinforce the idea that local unfolding is involved in enzyme catalysis and suggest a key role in the regulation of enzyme activity.