Aquaporin Z Protein

Anna Harnsberger '23 and Emma Becker '22

Contents:

I. Introduction

The Escherichia coli (E.Coli), contains an Aquaporin Z tetramer protein (Aqpz). It is a homotetramer protein involved in the facilitation of water movement across the plasma membrane of E. Coli. Aqpz is part of a family of proteins, aquaporins. Aquaporins facilitate transmembrane fluctuations of water or glycerol, the rapid transmembrane fluid fluctuations maintain homeostasis within the cell.

In prokaryotes, such as E. Coli, aquaporins help protect the cell from osmotic shock and rapid freezing.

The residues that line the monomeric channels are almost identical in their positions, except for the side chain of a highly conserved amino acid, Arginine-189 (Arg-189). Arg-189 takes on two distinct conformational orientations:

1)In one of the monomers, the guanidino group of Arg-189 is positioned toward the periplasmic vestibule and opens the channel. This allows for the binding of a water molecule through a tridentate H-bond.

2)In the other three monomers, the guandino group of Arg-189 bends over and forms an H-bond with carbonyl-oxygen of the Threonine (Thr-183), causing the channel to close.

The conformational state of Arg-189 has been found to mediate water permeability within the cell . 

II. General Structure

Aqpz is a , the natural tetramer structure of this protein reinforces the significance of oligemirzation. Though this protein is a tetramer, the individual monomers are the functional subunits of the protein. Tetramerization of the protein is caused by the energetically favorable four protomers, structural subunits of the .

III. Water Channels

The aquaporin channel of each monomer is relatively long and thin (a length of 28 angstroms and a diameter of 4 angstroms). It is formed by the packing of helices in two domains: and . The NPA region—asparagine (purple), proline (blue), and alanine (red)—is highlighted as an important area for water binding.

The two domains together form an amphipathic channel for waters to proceed single-file. The hydrophilic nature of the channel comes primarily from the carbonyls and amino groups on the peptide backbone that face the inside of the channel—together they hydrogen-bond to five water molecules at a time . The hydrophobic nature of the channel comes from a large number of phenylalanine, valine, and isoleucine. There are two conserved regions within the channel—the NPA region and the selectivity filter. The NPA region consists of two asparagine-proline-alanine motifs, one from each domain of the channel. The asparagine residues' side chain amino groups hydrogen-bond with the central water molecule, and their backbone carbonyl and amino groups hydrogen-bond with nearby alanine and valine backbones to increase stability . The proline and alanine residues of the NPA region hydrophobically clump together behind the asparagine residues.

The NPA is hypothesized to perform an important function for the aquaporin. As can be seen in , the water molecules are stacked in a narrow chain in the channel. Normally, single water molecules placed in a line should orient themselves dipole-to-dipole to form a wire, allowing protons to jump from molecule to molecule. However, the aquaporin prevents this from happening—the asparagine residue of the NPA region donates two hydrogen bonds to the oxygen atom of the central water (W5), locking it into a conformation where it can only donate H-bonds to other water molecules, never accept them . This preserves the electrochemical gradient in the channel.

The selectivity channel is the narrowest section of the channel, and is made up of a threonine, histidine, arginine, and phenylalanine residue . The three hydrophilic residues form a hydrogen-bonding triangle for the water molecule passing through opposite the hydrophobic phenylalanine. This small, amphipathic selectivity channel only permits water to pass through—in contrast, the glycerol transport aquaporin GlpF has a much larger and more hydrophobic channel to select for glycerol.

IV. Arginine-189, Conformation and Orientation

An important aspect of AqpZ is the closed and open state conformation, thermodynamic fluctuations of the Arg-189 side chain dictate the conformational state of the water channel. Within the four monomer subunits of AqpZ, one is found in the fully open state and the other three remain closed as a result of the displacement of Arg-189. The closed conformation of the three monomers obstucts the selective filter.

Arg-189 has a side chain that consists of a 3-carbon aliphatic straight chain, the distal end is capped by a guanidinium group. Within of the protein, the guanidinium group of Arg-189 is oriented parallel to the water channel. This orientation causes the 'A' monomer to adapt to the open-conformational state and enables a water molecule to be bound within the channel. Water molecules WA1 and WA2 are present within the channel. WA1 is hydrogen bonded to the NH2 of the R-189 gunaidino group, located the selective filter. WA2 is located within the selective filter and forms a with NE2 of Histidine (His-174), NE of Arg-189, and the main chain carbonyl-oxygen of Thr-183.

The other three monomers B, C, and D adapt to the closed-conformation state. This conformational change is a result of the Arg-189 guanidino group bending toward the carbonyl oxygen of Thr-183, forming a NH1-O hydrogen bond and closing up the selective filter. In , water molecule WB1 is hydrogen bonded to the carbonyl-oxygen of Thr-183 and Arg-189 NH1, located the selective filter. and have almost identical bonding of amino acids, and do not contain water molecules within their channels.

V. Applications

AqpZ is a bacterial analog to Aqp4, an aquaporin found in the human brain—specifically within astrocytes and tissues that are in contact with blood vessels or cerebrospinal fluid. Aqp4 is very important to the function of the nervous system—its malfunction is responsible for several diseases of the nervous system, such as epilepsy, brain edema, and lupus cerebritis (3). A possible link to Alzheimer's has also been explored—Aqp4 may affect calcium and potassium signalling in astrocytes, help with the removal of protein-waster beta amyloid from interstitial fluid, and influence neuroinflammation (2). Recently, Aqp4 has been found to be falsely identified as an antigen and targeted by IgG autoantibodies, resulting in the disorder Neuromyelitis Optica (NMO) that is similar to multiple sclerosis. Therefore, Aqp4 has been the target of therapeutics that seek to alter its permeability to water (3).

Because AqpZ is so similar in structure to Aqp4, it has been the target of interesting studies that attempt to explain Aqp4-related disorders. For example, Ren et al. found that AqpZ and Aqp4 have cross-immunoreactivity. The study produced immune mouse serum against bacterial AqpZ before testing it against Aqp4, and found that Aqp4 did react with the serum in the same way that AqpZ did (4). This implies that the similar homology between the two aquaporins can cause IgG to mistake Aqp4 for the bacterial AqpZ after an infection, and that immune response would result in the development of NMO in a patient. This information may also be useful in developing an animal model for NMO using NMO-inducing immune serum against AqpZ, since there currently is no animal model for testing therapeutics (4). Using AqpZ as a model for Aqp4 may serve medical research into many different disorders of the nervous system.

VI. References

(1)Jiang, J.; Daniels; B. V.; Fu, D. Crystal Structure of Aqpz Tetramer Reveals Two Distinct Arg-189 Conformations Associated with Water Permeation through the Narrowest Constriction of the Water-conducting Channel. J. Biol. Chem. 2006. 281, 454-560. doi: 10.1074/jbc.M508926200.

(2)Lan, Y. L.; Chen, J. J.; Hu, G.; Xu, J.; Xiao, M.; Li, S. Aquaporin 4 in Astrocytes is a Target for Therapy in Alzheimer's Disease. Curr Pharm Des. 2017. 23(33), 4948-4957. doi: 10.2174/1381612823666170714144844.

(3)Mangiatordi, G. F.; Alberga, D.; Trisciuzzi, D.; Lattanzi, G.; Nicolotti, O. Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future. Int J Mol Sci. 2016. 17(7), 1119. doi: 10.3390/ijms17071119.

(4)Ren, Z.; Wang, Y.; Duan, T.; Patel, J.; Liggett, T.; Loda, E.; Brahma, S.; Goswami, R.; Grouse, C.; Byrne, R.; Stefoski, D.; Javed, A.; Miller, S. D.; Balabanov, R. Cross-Immunoreactivity between Bacterial Aquaporin-Z and Human Aquaporin-4: Potential Relevance to Neuromyelitis Optica. J Immunol. 2012. 189(9), 4602-4611. doi: 10.4049/jimmunol.1200486.

(5)Savage, D. F.; Egea, P. F.; Robles-Colmenares, Y.; O'Connell, J. D.; Stroud, R. M. Architecture and Selectivity in Aquaporins: 2.5 A structure of Aquaporin Z. PLOS Biol. 2003. 1(3), e72. doi: 10.1371/journal.pbio.0000072.