The mutation G133D on Leishmania guyanensis AQP1 is highly destabilizing as revealed by molecular modeling and hypo-osmotic shock assay

Abstract

The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1−/− mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.