Structural Details of Human Aquaporin Regulation : Three stories of three aquaporins

Sammanfattning: Every cell is surrounded by a thin plasma membrane, protecting it from its surrounding. Specialized protein channels and transporters are embedded in this membrane, ensuring selective transport of molecules in and out of the cells. To maintain optimal hydration, water channels called aquaporins (AQPs) are present in every cell of our body. Thirteen isoforms of aquaporins exist in humans of which three, AQP2, AQP5 and AQP0, were studied in this thesis. The water passage through aquaporins needs to be very tightly regulated which happens by two distinct post-translational mechanisms. Either, the protein can be trafficked to the plasma membrane from storage vesicles when needed, like the case of AQP2 and AQP5. Alternatively, the channel might open and close based on the needs of the cell, which is a preferred way of regulation for AQP0. The need for regulation is signaled by a trigger – for example by changes in osmolarity and pH or by a hormone binding to its receptor. This signal is then carried out by binding to other regulatory proteins and/or by post- translational modifications, specifically phosphorylation.In this thesis we studied the structural details of three protein-protein interactions involved in regulation of human AQP2, AQP5 and AQP0 by trafficking or gating. The binding of AQP2 to lysosomal trafficking regulator- interacting protein 5 (LIP5) targets AQP2 to multivesicular bodies of the endosomal sorting pathway. We quantified the effect of phosphorylation on this interaction using microscale thermophoresis (MST) and we studied the binding interface using molecular docking, mutational studies and fluorescence quenching. Our results reveal that LIP5 binds AQP2 in a same way as it binds the ESCRT-III complex of the endosomal sorting machinery. We have identified residues important for the interaction and showed that AQP2 phosphorylation at specific sites located outside the LIP5 binding site impairs the binding to LIP5. Moreover, we have obtained an 8Å cryo-EM model of the complex in a phospholipid nanodisc, which confirms our previous results and shows that two molecules of LIP5 bind each AQP2 tetramer. This structure and the method used to obtain it serve as a stepping stone towards structure determination of other aquaporin complexes.For AQP5, the interaction with prolactin-inducible protein (PIP) is suggested to be important for its trafficking to the plasma membrane. We have characterized the interaction between the full-length proteins using MST and showed that it is mediated by the distal C-terminus and that one PIP molecule binds the AQP5 tetramer. In case of AQP0, the binding of calmodulin (CaM) upon increased intracellular calcium concentrations closes the channel. Using MST and a liposome-based water permeability assay, we showed that binding of CaM is inhibited by AQP0 phosphorylation at two different sites but that phosphorylation of a third site allows CaM to bind in a manner that keeps the channel open.Our studies give new insights into the role of protein-protein interactions and phosphorylation in two distinct AQP regulatory mechanisms, thereby significantly increasing our understanding of how cellular water permeability can be controlled in a tissue-dependent manner.