Crystal structure of human aquaporin 4 at 1.8 A and its mechanism of conductance

Abstract

Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood-brain and brain-cerebrospinal fluid interfaces of glial cells. Its function in cerebral water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-A crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel.

Fig. 1.

General features. (A and B) Monomer and tetramer views of hAQP4 in diagram representation. Brown and orange colors represent the N- and C-terminal pseudo 2-fold related portions. Water molecules are represented as red spheres, and glycerol molecules are shown as green sticks. (A) The side view of the monomer. Helices are labeled M1 to M8. (B) The tetramer viewed from the extracellular side down the crystallographic 4-fold symmetry axis. (C) The network of water molecules found at the intracellular side of the central pore. The central pore is at the crystallographic 4-fold symmetry axis, and is formed by the tetramer. The 2Fo-Fc density of the water molecules is shown in black, contoured at 1.2 σ. The backbone amides of Ser-188 and Gly-189 are colored yellow in diagram representation. Phe-195 is shown as brown stick and cyan surface. (D–F) Diagram representation of the C loop of all of the AQP X-ray structures solved to date. (D) E. coli GlpF (brown), archeal AqpM (magenta), spinach AQP SoPIP2;1 (blue), and PfAQP (green). (E) Rat AQP4 (yellow), human AQP4 (black), human AQP5 (red), E. coli AqpZ (cyan), bovine AQP0 (green), and bovine AQP1 (purple). (F) Comparison of the 3₁₀ helix of rat AQP4 (yellow) with the 2-turn helix of AqpM (magenta) and spinach AQP (blue). All structural renderings were made with PyMOL (http://www.pymol.org).

Fig. 2.

The conducting pore. The trace of the pore inner surface is shown in cyan. The selectivity filter residues, Arg-216 and His-201, are shown as sticks with surfaces in purple. The glycerol molecule is shown as green stick, and the water molecules in the channel are shown as red spheres. (B) Plot of the channel radius versus position along the pore for human AQP4, bovine AQP1 (bAQP1), and the P. falciparum AQP (PfAQP). Regions of the channel are labeled as extracellular vestibule, the selectivity filter (SF), the NPA motif, and the intracellular vestibule. The pore inner surface and its dimension are calculated using Hole 2.0 (51).

Fig. 3.

Electron density. Residues that form the wall of the pore are shown in sticks. Water molecules are shown as red spheres. The glycerol molecule is shown in green stick. The 2Fo-Fc density is shown in black, contoured at 1.2 σ. Positive F_o − F_c density is shown in green, contoured at 3 σ. There is no negative F_o − F_c density.

Fig. 4.

Comparison of the hydrogen bond network of the selectivity filter arginine of hAQP4, bAQP1, and GlpF. Protein C-alpha is shown in diagram representation. Residues of the selectivity filter and glycerol molecules are shown as sticks. Water molecules are shown as red spheres.

Fig. 5.

The NPA motifs. (A) Schematic representation of the hydrogen bonding network through the channels of hAQP4 and bAQP1. The distances are between heavy-atom to heavy-atom. (B) Stick representation of the NPA motifs. Distances that are too long to be a hydrogen bond are colored in red. (C) Plot of the MD simulations of hAQP4 from 4 different experiments. Details are described in Discussion.