Abstract
Aquaporin TIP2;1 is a protein channel permeable to both water and ammonia. The structural origin of ammonia selectivity remains obscure, but experiments have revealed that a double mutation renders it impermeable to ammonia without affecting water permeability. Here, we aim to reproduce and explain these observations by performing an extensive mutational study using microsecond long molecular dynamics simulations, applying the two popular force fields CHARMM36 and Amber ff99SB-ILDN. We calculate permeabilities and free energies along the channel axis for ammonia and water. For one force field, the permeability of the double mutant decreases by a factor of 2.5 for water and 4 for ammonia, increasing water selectivity by a factor of 1.6. We attribute this effect to decreased entropy of water in the pore, due to the observed increase in pore–water interactions and narrower pore. Additionally, we observe spontaneous opening and closing of the pore on the cytosolic side, which suggests a gating mechanism for the pore. Our results show that sampling methods and simulation times are sufficient to delineate even subtle effects of mutations on structure and function and to capture important long-timescale events, but also underline the importance of improving models further.
Highlights
Aquaporins are a family of transmembrane proteins found in cells of all types of organisms, from eukaryotes to bacteria and archaea
In the following we present results from 1 μs long simulations of four different systems: wild type TIP2;1, in the following referred to as TIP2;1, G194C, I185H and I185H × G194C, applying both CHARMM and Amber force fields
Since we expect the core of the channel to be stable, we consider it most likely that these events arise from modeling issues, such as the force field in combination with the starting structure
Summary
Aquaporins are a family of transmembrane proteins found in cells of all types of organisms, from eukaryotes to bacteria and archaea. In humans more than ten different aquaporins are expressed in the body, each with its own specific function These nano-sized pores reside in the hydrophobic membrane where they provide a path for water to efficiently pass through. One expects ammonia to be able to fit through narrow constrictions just like water and to readily hydrogen bond both to water and to polar sites of the pore. Both mammalian and plant aquaporins have been reported to be permeable to ammonia, at least to some degree, based on experiments using expression systems of yeast or egg cells of frog[1,7] It was suggested based on this structure that the SF should be extended to include a fifth residue, H131, with the motivation that it sterically enforces the special position of R200 and that it interacts with water molecules entering the pore
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