Human Aquaporin-1: Furosemide Docking and Protein-Protein Interactions

DORR R; ROSI P; OZU M; COSTA ALMAR F; TORIANO R

Sierra de la Ventana

Congreso; XLIII Reunión Anual 2014; 2014

Sociedad Argentina de Biofísica

P { margin-bottom: 0.21cm; } Human Aquaporin-1: Furosemide Docking and Protein-Protein Interactions Dorr, RA1; Rosi, P2; Ozu, M1; Costa Almar, F1; Toriano, R1 1Lab. de Biomembranas-IFIBIO Houssay-UBA-CONICET; 2DQIAyQF-FCEN-UBA In our previous work we showed that the water permeability of human aquaporin-1 (hAQP1) is specifically inhibited by the presence of intracellular furosemide and modulated by the tension of the plasma membrane. These data were obtained in the Xenopuslaevis oocyte membrane expressing water channels. Now we use different docking protocols to locate sites of inhibition and modifiers of membrane tension. The first strategy was to perform a blind docking (with AutoDock) against the monomeric structure of hAQP1 obtained from electron-crystallographic data (1FQY PDB code). The results indicate that furosemide binds to both cytosolic and outside areas of the channel, but never at sites in the transmembrane region. Coincidentally with the in vitro results, the cytosolic region was more favored than the extracellular binding region. To evaluate the influence of protein conformational changes, we did an unrestricted α-carbon molecular dynamic simulation (50ns in explicit aqueous solvent, repeating docking protocol for 10 frames separated by 5ns each). A significant change in the angle of the α-helices and a pore occlusion (quantified with PoreWalker server) could be observed. Under these conditions, the furosemide binding sites are lost quickly. To simulate hAQP1 in an environment that resembles protein included in a lipid bilayer, α-carbon fixation was used. In such condition, the distinction between furosemide binding sites remains. Once again the intracellular binding site was favored. Based on published data in which the presence of Na+ channel ENaC modifies the membrane tension, we also studied the co-expression of hAQP1 and ENaC in Xenopus oocytes. The in vitro data plus the possibility of formation of a protein-protein interaction (that was found using the ClusPro server) lead us to hypothesize a physiological and morphological interaction between the two proteins.