Aquaporin-1
Created by So Hyun Park
Aquaporin-1(AQP1) (PDB ID=1J4N) from Bos taurus is a water transport channel which selectively facilitates the rapid transport of water across the cell membranes due to osmotic gradients (1). The aquaporin is a membrane channel protein whose presence is critical for life-sustaining of most organisms because water is essential for life. Approximately 70% of human body is composed of water, so the appropriate balance of water within different compartments is required to maintain solute/fluid balance throughout the body. Therefore, the control of membrane water permeability by aquaporin is the primary condition for life (6).
There are ten families of water channels (AQP0-AQP9) in mammals and these channels are involved in numerous physiological processes including neuro-homeostasis, digestion, regulation of body temperature, reproduction and renal water conservation (1). Although all aquaporin families have ability to conduct water within different ranges, some of them can transport not only water but also other solutes such as monosaccharides and glycerols (4). AQP1 was first discovered as an integral membrane protein in erythrocytes and renal proximal tubules where it acts as constitutively open, bidirectional water selective channel and is broadly expressed in the plasma membranes of various water-permeable epithelial and endothelial cells (5). AQP1 is highly specific to the water because other solutes and ions including protons should not cross the membrane freely through the pore (2). According to ExPasy, the relative molecular weight of AQP1 is 28,000 Da, and its isoelectric point (pI) is 6.65 (1).
The structure of aquaporin-1 is deeply related to its physiological function. AQP1 associates as a homotetrameric functional unit with each monomer presenting an independent water pore. Each monomer has six membrane-spanning α-helices (M1-M2, M4-M6, M8) arranged in a right-handed bundle. This structure surrounds two membrane-inserted but non-membrane-spanning α-helices (M3, M7) which are the major portions of the pore. According to the sequence analysis, a great homology exists among AQP superfamily. Each half of the sequence contains a NPA (Asparagine, Proline, Alanine) motif, which is highly conserved throughout the AQP superfamily (1, 5, 7). For this reason, general folding pattern of glycerol uptake facilitator protein (GlpF) of E.coli shows great topological similarity to that of AQP1 (1).
The short amino terminus of aquaporin-1 is placed on the cytoplasmic side of the cell membrane and followed by two membrane-spanning α-helices (M1, M2). A membrane-inserted loop including the NPA motif leads into the non-membrane-spaning α-helix (M3). M3 helix enters the cytoplasm and turns back to transmembrane helix M4. This concludes the N-terminal half of the AQP1 structure which is on the extracellular side of the cell membrane. The other half of the AQP1 structure is almost the exact repeat of the reversed first half (1).
The dumbbell-like shape of the pore of aquaporin-1 structure is comprised of three elements: extracellular vestibule, cytoplasmic vestibule and extended narrow pore containing constriction region which acts as a selective filter. Wall of the selective channel is showing amphipathic nature consisting of roughly 50% of hydrophobic and 50% of hydrophilic residues. Hydrophilic side of the channel creates the water transport pathway by presenting the chemical groups which have a crucial role in displacing waters. However, M3 and M4 α-helices are not considered as parts of the pore. Loop region of the monomer face and N- terminal/C-terminal residues of the cytoplasmic face form the vestibule mouth (1).
Sharp and steep crossing angles of the monomer helices help the extracellular and cytoplasmic vestibules to form. Mouth diameter of the cone shaped extracellular vestibule is around 15Å in pore size which is based on the van der Waals radii. This diameter of the extracellular vestibule narrows to 2.8 Å over a distance of about 20 Å. Polar residues are dominantly located on the surface of the extracellular vestibule and few charged and polar groups are present on the extended loop region of the solvent-exposed backbone. Cytoplasmic vestibule is the last 8-10 Å region of the channel. Its mouth is approximately 15 Å wide in diameter. Comparing to the shape of extracellular vestibule, it is more conical in shape. Since the population of polar residues drastically increases at this region, these residues form more hydrophilic region than that of GlpF (1).
Three residues define the constriction region of the aquaporin-1 pore: Arg-197, His-182, and Phe-58. These residues are specifically conserved through aquaporin superfamily and responsible for water specificity. At this region, His-182, Arg-197 and Cys-191 form hydrophilic face of the pore discussed earlier. The bulk group of Arg-197 is sticking upwards parallel to the pore axis and the imidazole ring of His-182 is fully reached into the pore. Phe-58 arranges the hydrophobic face which is opposite to the hydrophilic face of this constriction region (1).
Four bound waters present inside the aquaporin-1 selectivity filter. The locations of these water molecules correspond to the hydrophilic nodes reported by the hydrophobicity profile of the residues aligning the filter. First water closest to the extracellular side is making hydrogen bonds with ε2 nitrogen of His-182 and the carbonyl oxygen of Gly-192. Second and third water molecules are hydrogen-bonded to the δ2 nitrogen of Asn-194 and δ2 nitrogen of Asn-78 respectively. The last water molecule placed nearest to the cytoplasmic side of the filter forms hydrogen bonds with His-76 and Ala-75 (1).
Physiological function of aquaporin-1 is to facilitate passive permeation of water across the cell membranes in accordance with the osmotic gradients. AQP1 facilitates water transport at a rapid rate of 3x109 s-1 per monomer channel with low activation energy comparable to that of self-diffusion in bulk water (2, 8). AQP1 transports water exclusively and does not permeate ions including protons. However, some aquaporins known as aquaglyceroproins transport both water and glycerol. Bacterial glycerol facilitator (GlpF) is the example of aquaglyceroproins (8). The highly specific and selective nature of AQP1 is important for both free water permeation through the channel and proton blockage. Especially, it is crucial for the kindney to reabsorb the water from glomerular filtrate while excreting the acid (6).
The selectivity for water is mainly governed by three criteria: size restriction, electrostatic repulsion and water dipole reorientation (6). The size limit of constriction region is around 2.8 Å. As a result, the effective diameter of the water needs to be reduced by shedding waters of hydration in order to diffuse across the constriction region. An adequate amount of hydrogen bond forming froups in AQP1 replace the primary hydration shell interaction and allow water molecule to move across the constriction region. His-182, Arg-197, Gly-190, Cys-191 and Gly-192 are responsible for the bond forming groups of AQP1. This explains why the transport of glycerol and large size molecules through AQP1 is unlikely (1). Secondly, the dipole moments of the helices in the pore region of AQP1 contribute to the positive electrostatic repulsion which results in a high dielectric barrier that repels ions such as protons (2). Lastly, water dipole disorientation by two short pore helixes (M3, M7) containing NPA motif interrupt the hydrogen bonding interaction of water and prevent the proton conductance (6). Thus, only water molecules can pass through the pore of AQP1 with a minimal energy barrier.
Although, size restriction, electrostatic repulsion and water dipole reorientation are the main contributing factors for solute selection and water permeation, there has been a study regarding the importance of His-76 and Val-155 in aquaporin-1. Depending on the orientation of the side chain of His-76, both His-76 and Val-155 can act as a valve to control the water conductance. The separation distance between His-76 and Val-155 is positively proportional to the water permeation. Water flow occurs when His-76 makes separation distance of 5.5-6.0 Å with Val 155 (5).
Considering the importance of water transport in physiological processes in the body, aquaporin family can cause the various diseases involving fluid transport disorder such as brain edema, lung edema, nephrogenic diabetes inspidus (NDI), dry eye, and congestive heart failure. For example, haman AQP0 which is the intrinsic protein of lens fiber cells has been identified to be responsible for the inherited cataracts. Individuals who lack human AQP1 have problems with mild renal concentration defects and water deprivations even though they were usually not aware of their physical limitations. Mutation of Arg-187 of human AQP2 is suspected to be involved in NDI. In 1993, Cys-189 residue of human AQP1 was identified as the target of the mercury metal ions which prevent the membrane transportation of water. Therefore, understanding of atomic structure and molecular dynamics of AQP1 is biologically significant because it can not only pave a novel way to solve human clinical disorders but also provide insights to the drug design (6).
The ligand component for aquaporin-1 is B-nonylglucoside. Three nonlyglucoside detergent molecules are located on the surface of the monomer contacting with the extracellular leaflet of the lipid bilayer (1). These ligands were used for crystallization.
Crystal structures of btAQP1 from bovine red blood cells were grown using the sitting-drop crystallization technique. The entire btAQP1 was concentrated to 20 mg/ml in 20 mM Tris-HCl buffer pH 7.5 and mixed with 1:1 volume ratio of 20% PEG monomethyl ether 550 and 10 mM Tris-HCl pH 7.5 solutions. Then, the structure was determined by X-ray crystallography at 2.2 resolution at which the functional unit of btAQP1 structure is most clearly represented. For example, the molecular position of water inside the transmembrane channel pathway can be visualized (1).
The secondary structure of aquaporin-1 is composed of 42-48% alpha helices and little or no beta sheets. Some random coils are displayed in AQP1. Alpha helices have tilted transmembrane orientation of 21-27 degrees (9). Six membrane-spanning α-helices (M1-M2, M4-M6, M8) make up the right hand bundle of the monomer and two membrane-inserted but non-membrane-spanning α-helices (M3, M7) compose the majority of the water pore (1,9). The structure of aquaporin-1 does not have binding sites of substrates, products, metal ions, or prosthetic groups.
The Glycerol uptake facilitator protein and the human aquaporin-5 were compared to the AQP1 to better understand how the structure of AQP1 dictates its function. For this purpose, the data from the Dali Server and the PSI-PLAST searches were analyzed. The purpose of Dali Server search was to compare protein structures in three-dimensions by evaluating E-scores. Additionally, PSI-BLAST search detected distant evolutionary relationships between the proteins by assigning corresponding E-value.
Glycerol uptake facilitator protein (pdb ID=1LDF) from Escherichia coli has an approximate 69% sequence similarity to aquaporin-1 and shows nearly identical topological folding pattern. The structural weight of GlpF is 30,532.44 Da which is 10% heavier than that of AQP1. The results of DALI (Z=27.7, rmsd=2.1) and protein blast (E=6e-14) searches show that GlpF has both primary and tertiary similarities to AQP1 (10). The associated ligand components of GlpF are B-octlyglucoside, glycerol, and magnesium ion. Structural differences provide an explanation of how these channels select their substrates.GlpF has glycine at position 191; whereas AQP1 has histidine at position 182. This replacement makes the additional space for phenylalanine to substitute the Cys-191 of AQP1 and significantly increase the size of the constriction region. These two substitutions in residues alter the polar environment of constriction region and result in enhanced channel interactions with the hydrophobic backbone of glycerol. Overall, these two substitutions are responsible for the two significant characteristic nature of the constriction region of the GlpF which are increased size and hydrophobicity (1).
There is another mechanism explaining how GlpF facilitates permeation of relatively larger glycerol molecule while preventing passage of relatively smaller water molecules. The selectivity filter of AQP1 extends for a single amino acid, and it is located in the middle of the membrane and shows a fine-tuned dipole inversion in the pore region so only small molecules with a large dipole moment such as water can pass through the dipole inverted region rapidly (8). However, this orientation pattern does not exist in GlpF. Instead, arginine side chains of GlpF forms hydrogen bonds with two hydroxyl groups of the glycerol whereas two aromatic side chains of Trp-48 and Phe-200 pack carbon backbone of glycerol against a hydrophobic wedge. This interaction allows the glycerol molecules to enter the selectivity filter of GlpF while excluding other linear polyols (7).
The human aquaporin-5 (hsAQP5) (PDB ID=3D9S) from Homo sapiens has around 95% structural similarity to bovine Aquaporin-1 (btAQP1). The structural weight of hsAQP5 is 114,165 Da which is four times heavier than that of btAQP1. The results of DALI (Z=35.0, rmsd=1.9) and protein blast (E=6e-68) searches show that hsAQP5 has high structural similarities to btAQP1 (10). Associated ligand component of hsAQP5 is O-[(S)-{[(2S)-2-(hexanoyloxy)-3-(tetradecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-D-serine. Similar to btAQP1, hsAQP5 facilitates the water flow across the cellular membrane while preserving ion concentration gradients in human body. The hsAQP5 has a fundamental physiological role in human body by maintaining the water homeostasis within the various cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, and the inner ear. Another notable feature of human aquaporin including hsAQP5 is the posttranscriptional modification of certain isoforms in addition to the usual tissue-specific regulations at transcriptional level. However, the detailed molecular mechanism of this regulation remains unidentified (11).
The btAQP1 contains a bulky Phe-176 residue near the center of the membrane which prevent the lipid insertion. In contrast, the hsAQP5 has a central pore cavity filled with lipid due to the compact Leu-167 which corresponds to the Phe-176 of btAQP1. In addition, conserved glycine residue of loop D creates cytosolic entrance to the central pore in btAQP1, but in hsAQP5, loop D is hydrogen bonded to the neighboring promoter thus adopting different conformation. The occlusion of the central pore by lipid is the distinguishing structural feature of the hsAQP5 compare to other mammalian AQPs including btAQP1. Due to this occlusion, the hsAQP5 tetramer is slightly less stabilized and has a reduced ability to transport gas molecules and ions compared to btAQP1 (11).