Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport.

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases.