Hydroxide and proton migration in aquaporins

Understanding how water and ions move through membrane proteins remains a central challenge in biophysics. While aquaporins are known to selectively transport water, the mechanisms governing hydroxide and proton migration remain unclear. Prior research has shown that aquaporins form single-file water chains, but the role of hydrogen bonding and electrostatic interactions in ion movement is less established. This gap motivated further investigation into how structural motifs and electrostatic dipoles influence ion migration. No prior work had resolved how conserved motifs like Asn-Pro-Ala affect hydroxide and proton movement. The need to clarify these interactions is critical for understanding selective ion transport in aquaporins. Existing models suggest that water reorientation is linked to proton transfer, but the exact sequence and influence of protein residues remain debated. This paper addresses the unresolved question of how conserved residues and macrodipoles regulate hydroxide and proton migration in aquaporins.

The study aimed to investigate the migration of hydroxide and proton ions through the Aquaporin GlpF from Escherichia coli using ab initio simulations. The specific problem addressed is how structural motifs and electrostatic interactions influence the movement of these ions. The motivation stems from the need to clarify the role of conserved residues like Asn-Pro-Ala and macrodipoles in ion transport. Previous studies lacked detailed insights into the dynamic behavior of water and ions within the channel. This work seeks to fill that gap by examining the reorientation of water molecules and the electrostatic coupling with conserved macrodipoles. The study also explores how protonation and deprotonation events affect ion migration. By focusing on the periplasmic and cytoplasmic half-channels, the authors aim to distinguish between different proton transfer mechanisms. The ultimate goal is to provide a mechanistic framework for how hydroxide and proton ions move through aquaporins.

The researchers employed ab initio Car-Parrinello molecular dynamics simulations to model hydroxide and proton migration in Aquaporin GlpF. These simulations allowed for the tracking of water-water hydrogen bonds and the polarization of water molecules along the channel axis. The study focused on the structural features of the protein, including conserved Asn-Pro-Ala motifs and oppositely aligned macrodipoles. The simulations captured the reorientation of water molecules during deprotonation events. The movement of hydroxide ions was analyzed in relation to the channel's electrostatic environment. The periplasmic and cytoplasmic half-channels were examined separately to compare proton transfer mechanisms. The study also evaluated the role of fourfold coordination in stabilizing hydroxide ions at specific motifs. Finally, the simulations provided insights into the influence of macrodipoles on hydroxide transition from the channel vestibules into the lumen.

The simulations revealed that Aquaporin GlpF stabilizes a bipolar single file of water molecules. This single file features a contiguous set of hydrogen bonds with varying polarization along the channel axis. Deprotonation of the water chain promotes reorientation of water molecules, enabling hydroxide migration. The hydroxide ion rapidly migrates by sequentially accepting protons from neighboring water molecules. The conserved Asn-Pro-Ala motifs stabilize the hydroxide ion through fourfold coordination. The hydroxide ion is immobilized at these motifs rather than being attracted to a channel-lining arginine residue. Electrostatic coupling with two oppositely aligned macrodipoles influences hydroxide transition from the channel vestibules into the lumen. The macrodipole's negative poles appear to prevent hydroxide ions from entering the inner vestibules. Water protonation in the lumen facilitates reorientation and subsequent proton expelling. In the periplasmic half-channel, proton expelling occurs via the Grotthuss mechanism. Protonation in the cytoplasmic half-channel suggests wire-breakage at the Asn-Pro-Ala motifs. Protons are diffusively rejected as (H5O2)+ in this region.

The authors propose that structural motifs and electrostatic interactions regulate hydroxide and proton migration in Aquaporin GlpF. The study suggests that the Asn-Pro-Ala motifs provide fourfold coordination, stabilizing hydroxide ions. The conserved macrodipoles influence hydroxide transition by electrostatic coupling, preventing ions from entering the inner vestibules. The findings indicate that water reorientation and proton transfer are linked to the polarization of water molecules. The periplasmic half-channel supports proton expelling via the Grotthuss mechanism. In contrast, the cytoplasmic half-channel involves wire-breakage at the Asn-Pro-Ala motifs. The study supports the idea that protonation and deprotonation events are essential for ion migration. The results align with the hypothesis that aquaporins use a combination of structural and electrostatic features to regulate ion movement. These conclusions are based on the observed behavior of water and ions in the simulations.