A conserved H-bond network in human aquaporin-1 is necessary for native folding and oligomerization.

Aquaporins (AQPs) are α-helical transmembrane proteins that conduct water through membranes with high selectivity and permeability. For human AQP1, in addition to the functional Asn-Pro-Ala motifs and the aromatic/Arg selectivity filter within the pore, there are several highly conserved residues that form an expansive hydrogen-bonding network. Prior solid-state nuclear magnetic resonance studies and structural conservation analysis have detailed which residues may be involved in this network. We explored this network by mutating the sidechains or backbones involved in hydrogen-bonding, generating the following mutants: N127A, V133P, E142A, T187A, R195A, S196A. The fold and stability of these mutants were assessed with attenuated total reflection Fourier transform infra-red spectroscopy coupled with hydrogen/deuterium exchange upon increasing temperature. We found that replacement of any of the chosen residues to alanine leads to either partial instability or outright misfolding at room temperature, with the latter being most pronounced for the N127A, V133P, T187A, and R195A mutants. Deconvolution analysis of the amide I band revealed considerable secondary structure deviations, with some mutants exhibiting new random coil and β-sheet structures. We also found that some of these mutations potentially disrupt the oligomerization of human AQP1. BN-PAGE and DLS data provides evidence towards the loss of tetramers within most of the mutants, meanwhile only the S196A mutant retains tetrameric organization. The molecular dynamics simulation of the wild-type, and the N127A, E142A, and T187A mutants show that these mutations result in major rearrangements of intra- and inter-monomer hydrogen-bond networks. Overall, we show that specific point mutations that perturb hydrogen-bonding clusters result in severe misfolding in hAQP1 and disruption of its oligomerization. This data provides valuable insight into the structural stability of human aquaporin-1 and has implications towards other members of the AQP family, as these networks are largely conserved among a variety of human and non-mammalian AQP homologs.