Modulating O2 Permeability of the Central Pore of RH50 by in Silico Site Directed Mutagenesis

Abstract

The Rhesus (Rh) family of integral membrane proteins exhibit ubiquitous expression among animals and their structural homology to ammonium transport (Amt) proteins suggests their function as ammonia/ammonium transporters. They have also been implicated in facilitating permeation of gaseous species, such as dioxygen and carbon dioxide, across the cell membrane. Rh50 proteins are expressed in erythrocytes, as well as in epithelial tissues of organs such as the kidneys where critical nitrogen processing occurs. Using the solved Rh50 structure from bacterial homologue NeRh50 found in Nitrosomonas europaea, we employed a combination of molecular dynamics and free energy approaches including implicit ligand sampling, explicit ligand sampling, umbrella sampling, and in silico mutagenesis to characterize O2 permeation pathway, mechanism, and energetics. Free-energy calculations reveal high-energy barriers both in the monomeric pore as well as the pore formed in the trimerization domain similar to those previously reported for CO2 and NH3. Based on the the free-energy calculation results, we identified key amino acids potentially responsible for the high barrier. Bioinformatic analysis of these sites shows that some of the residues lining the high-energy region of the pore exhibit a high degree of conservation only for members of the Rh50 family, with the exception of NeRh50. Residues Thr199 in Arg202, which are localized near the pore opening, exist as hydrophobic amino acids in the protein's eukaryotic counterparts and Leu206 in NeRh50 presents itself primarily as an isoleucine. Mutation of these residues to the conserved amino acids results in a reduction in the energy barrier. This suggests a permeation pathway for gaseous species not observable in the native NeRh50 structure which can be verified by functional characterization of the mutants.