Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases.

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1'). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1' folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

The transported cations impose differences in the thermostability of the gastric H,K-ATPase. A kinetic analysis.

This work analyses the thermostability of a membrane protein, the gastric H,K-ATPase, by means of a detailed kinetic characterization of its inactivation process, which showed to exhibit first-order kinetics. We observed parallel time courses for the decrease of ATPase activity, the decrease of the autophosphorylation capacity and the loss of tertiary structure at 49 °C. Higher temperatures were required to induce a significant change in secondary structure. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the decrease of the residual activity after heating at that temperature, proves the irreversibility of the inactivation process. Inactivation proceeds at different rates in E1 or E2 conformations. The K+-induced E2 state exhibits a lower inactivation rate; the specific effect is exerted with a K0.5 similar to that found at 25 °C, providing a further inkling that K+ occlusion by the H,K-ATPase is not really favoured. Increasing [H+] from pH 8 to pH 7, which possibly shifts the protein to E1, produces a subtle destabilizing effect on the H,K-ATPase. We performed a prediction of potential intramolecular interactions and found that the differential stability between E1 and E2 may be mainly explained by the higher number of hydrophobic interactions in the α- and ß-subunits of E2 conformation.

Zoledronate repositioning as a potential trypanocidal drug. Trypanosoma cruzi HPRT an alternative target to be considered.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 7 million people worldwide. Considering the side effects and drug resistance shown by current treatments, the development of new anti-Chagas therapies is an urgent need. T. cruzi hypoxanthine phosphoribosyltransferase (TcHPRT), the key enzyme of the purine salvage pathway, is essential for the survival of trypanosomatids. Previously, we assessed the inhibitory effect of different bisphosphonates (BPs), HPRT substrate analogues, on the activity of the isolated enzyme. BPs are used as a treatment for bone diseases and growth inhibition studies on T. cruzi have associated BPs action with the farnesyl diphosphate synthase inhibition. Here, we demonstrated significant growth inhibition of epimastigotes in the presence of BPs and a strong correlation with our previous results on the isolated TcHPRT, suggesting this enzyme as a possible and important target for these drugs. We also found that the parasites exhibited a delay at S phase in the presence of zoledronate pointing out enzymes involved in the cell cycle, such as TcHPRT, as intracellular targets. Moreover, we validated that micromolar concentrations of zoledronate are capable to interfere with the progression of cell infection by this parasite. Altogether, our findings allow us to propose the repositioning of zoledronate as a promising candidate against Chagas disease and TcHPRT as a new target for future rational design of antiparasitic drugs.

The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1-E2 conformational changes assessed by eosin fluorescence measurements.

H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1-E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K â†’ E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that-during dephosphorylation-the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Šresolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.