Moss FJ et al. (2020), Aquaporin-7: A Dynamic Aquaglyceroporin With Gr...

XB-ART-57202

Front Physiol 2020 Jun 30;11:728. doi: 10.3389/fphys.2020.00728.

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Aquaporin-7: A Dynamic Aquaglyceroporin With Greater Water and Glycerol Permeability Than Its Bacterial Homolog GlpF.

Moss FJ , Mahinthichaichan P , Lodowski DT , Kowatz T , Tajkhorshid E , Engel A , Boron WF , Vahedi-Faridi A .

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Xenopus oocytes expressing human aquaporin-7 (AQP7) exhibit greater osmotic water permeability and 3H-glycerol uptake vs. those expressing the bacterial glycerol facilitator GlpF. AQP7-expressing oocytes exposed to increasing extracellular [glycerol] under isosmolal conditions exhibit increasing swelling rates, whereas GlpF-expressing oocytes do not swell at all. To provide a structural basis for these observed physiological differences, we performed X-ray crystallographic structure determination of AQP7 and molecular-dynamics simulations on AQP7 and GlpF. The structure reveals AQP7 tetramers containing two monomers with 3 glycerols, and two monomers with 2 glycerols in the pore. In contrast to GlpF, no glycerol is bound at the AQP7 selectivity filter (SF), comprising residues F74, G222, Y223, and R229. The AQP7 SF is resolved in its closed state because F74 blocks the passage of small solutes. Molecular dynamics simulations demonstrate that F74 undergoes large and rapid conformational changes, allowing glycerol molecules to permeate without orientational restriction. The more rigid GlpF imposes orientational constraints on glycerol molecules passing through the SF. Moreover, GlpF-W48 (analogous to AQP7-F74) undergoes rare but long-lasting conformational changes that block the pore to H2O and glycerol.

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Species referenced: Xenopus
Genes referenced: aqp1 aqp7 bag3 myc nps
GO keywords: cellular response to osmotic stress

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	FIGURE 1. The dependence of Pf assays on solute composition in oocytes expressing AQPs. (A) Osmotic swelling assays. Here we lower osmolality to 105 mOsm by transferring oocytes to our ND51 solution, or HEPES-buffered solutions (pH 7.50) in which the only other solutes are NaCl or glycerol. (B) Osmotic shrinkage assays. After the oocytes in panel (A) are re-equilibrated at 195 mOsm overnight in OR3 media, we raise osmolality to 270 mOsm by transferring them to ND96 supplemented with mannitol, or to HEPES-buffered solutions (pH 7.50) in which the only other solutes are NaCl or glycerol. Bars represent mean Â± S.E.M. The number of replicates are displayed at the base of each bar in panel (A), and are the same in panel (B). We harvested the oocytes from three frogs to perform these experiments. Statistics are performed as described in section âMaterials and Methods.â The symbol â*â denotes a significant difference compared to the H2O control injected oocytes in the same buffer. The symbol ââ â denotes a significant difference compared to both oocytes from the same cRNA injection type compared to NaCl buffer and compared to the H2O control injected oocytes in the same buffer.
	FIGURE 2. Normo-osmotic swelling of AQP7 and GlpF (Â± AQP1) expressing oocytes. (A) Oocyte swelling rates for WT AQP7 channels expressed alone or co-expressed with AQP1 as [glycerol]o increased in a normo-osmotic buffer. (B) Oocyte swelling rates for WT GlpF expressed alone or co-expressed with AQP1 as [glycerol]o in increased in a normo-osmotic buffer. All data points represent mean Â± S. E. M. These data and the number of oocytes and oocyte preparations/cRNA injections used to generate each point are detailed in Supplementary Tables S1â€“S3.
	FIGURE 3. AQP7 Structure. (A) Van-der-Waals surface representation of the AQP7 glycerol/water channel comprising the extracellular vestibule, the closed selectivity-filter (SF, shaded yellow), the NPA constriction (shaded purple) and the cytosolic vestibule. The view into the channel has been generated by omitting half of the protein. Channel lining residues are shaded peach. (B) Side view of one of the four AQP7 monomers, showing glycerol channel occupancy and detailed views of the NPA (NAA/NPS arrangement) and SF region. Due to the high symmetry (F4132) and the presence of non-crystallographic symmetry, side-chain electron density features are resolved at near atomic detail. Selected 2Fo-Fc maps highlight details of resolved large side chains for (i) the cytosolic loop B residues R106, V107, W109 and R110 and (ii) three glycerol molecules bound in the monomeric pore. (C) 2Fo-Fc map highlighting details of the SF region residues F74, Y223, and R229. Because residue G222 does not participate in the SF, it is not shown for clarity. (D) 2Fo-Fc map highlighting details of the NPA region residues N226, P227, S228, N94, A95, and A96. At early stages of refinement and model building, when we were employing 4-fold symmetry-averaged maps, we were able to observe a clear density, consistent with glycerol at the NPA of all 4 monomers. Thus, a glycerol is present at the NPA, albeit at low occupancy. In contrast to GlpF, no ligand electron density is observed at the SF. Two of the four monomers (including the one shown here) contain an additional glycerol molecule bound at the cytoplasmic surface (CS) region of the monomeric channel. Loop B and glycerols in panel B are contoured at 1.5 Ïƒ, SF residues in panel C are contoured at 2 Ïƒ, and NPA residues in panel (D) are contoured at 1.5 Ïƒ.
	FIGURE 4. Structures of the EV, SF and NPA regions. (A) Location EV: View of the monomeric extracellular channel entrance of AQP7 (cyan) with a lateral shift (5.5 Ã…) in the position of the resolved glycerol [center: AQP7 (red-cyan), shifted right: GlpF (orange)]. We depict the narrowing of the entrance by overlaying the protruding residues of GlpF (D130, K33, L32 and Q41) and AQP7 (A153, V59, M58, and Y67). Note the inward and outward facing conformations of Y67. (B) Location SF: Overlay of Selectivity filter (SF) region for AQP7 and GlpF formed by two aromatic residues (Y223, F74 in AQP7), positioned at a right angle to each other, forming a hydrophobic wedge opposite from R229 and G222. G222 points away from the SF, and does not participate in the restriction. We therefore omit G222 from the figure for clarity. The glycerol molecule (transparent orange spheres) shown at this location is only present in the GlpF SF; the AQP7 SF is unoccupied. A glycerol molecule (orange spheres) at this location is present only in GlpF. (C) Details of the van der Waals packing arrangement of the NAA-NPS motifs involving a slight out of plane rotation of the NPS motif. The glycerol molecule at this location is not displayed in this panel for clarity and because of partial disorder and low occupancy, as documented in the PDB submission. A polar contact is observed between S228 and S123 of Helix 2. The distance unit of the annotated dashed lines is Ã….
	FIGURE 5. Glycerol translocation along through AQP7 and GlpF. (A) Free energy (Î”G) profiles of glycerol partitioning along the pores of AQP7 calculated from the umbrella sampling simulations. (B) Orientation (P1) profiles of the molecule along the AQP7 (i.e., P1 fluctuates between +1 and âˆ¼ â€“1 and Î¸ from âˆ¼0Â° to âˆ¼180Â°). (C,D) Î”G and P1 profiles of glycerol partitioning in GlpF. The profiles along the individual pores are shown as individually colored thin lines. Î¸ is the angle between the vector connecting the first and last carbon atoms of the glycerol molecule and the membrane normal (approximately the pore axis). P1 is âˆ¼ Â± 1 when the orientation of the molecule is parallel to the pore axis, and <P1> is âˆ¼0 when it is isotropic. (E) Overall conformational dynamics of AQP7 versus GlpF characterized by time-averaged RMSD profiles calculated over all monomeric channels and over all 3 flooding simulations for each protein. The NPA and SF regions are highlighted in vertical purple and brown bars, respectively. Residues of helical regions involved in glycerol permeation are highlighted in horizontal green bars. (Fâ€“I) MD snapshots depicting glycerol transition through the SF region. The SF is centered at z = âˆ¼ 7.5 Ã…. F74 of AQP7 (purple) changes its conformations as the molecule transits between the extracellular vestibule and the NPA region. It is equivalent to W48 in GlpF. Also, Y223 of AQP7 is replaced by a phenylalanine (F200) in GlpF. (Jâ€“M) Conformations of the SF residues (F74 and Y223 of AQP7, and W48 and F200 of GlpF) as function of glycerol moleculeâ€™s position calculated from the umbrella sampling simulation, characterized by their N-CÎ±-CÎ²-CÎ³ (Ï‡1) dihedral angles.
	FIGURE 6. Water and glycerol permeabilities of AQP7 and GlpF. (A) Channel-dependent osmotic water permeability (Pf) for AQP7 and GlpF constructs. Pf for positive control AQP1 oocytes is 2.01 Â± 0.10 Ã— 10â€“3 cm/s (n = 81). We harvested oocytes from three frogs to perform these experiments. (B) AQP7 or GlpF in the surface fraction from biotinylated oocytes detected by western blot using a c-myc primary antibody. The predicted molecular weights of full-length myc-HIS tagged AQP7 and GlpF constructs are 39.8 and 32.4 kDa, respectively. AQP7 construct bands actually run in the 10% Bis-Tris gels at âˆ¼32.5 kDa. GlpF constructs run as a doublet of 25 and 22 kDa. These smaller than expected bands reflect differing degrees of N-terminal truncation because the epitope for the primary antibody is at the C-termini of all constructs. Protein bands are aligned with the corresponding Pf* bar in panel (A). AQP7 and GlpF samples are analyzed on separate blots denoted by a frame around each blot image. Displayed to the side of each blot are the molecular weight markers (Other example blots are in Supplementary Figure S8). (C) 10-min 3H-glycerol uptakes for AQP7 and GlpF constructs in an ND96 solution with 90 mM total [glycerol]0 replacing 45 mM NaCl. For both panels (A,C), bars represent mean Â± S.E.M. and the number of replicates is at the base of each bar, or in parentheses above the bar. â€ denotes Pf* for GlpF WT, which is much smaller than AQP7 WT. â€¡ in panel C denotes that uptake for both AQP7 WT and GlpF WT are significantly above the H2O control (p = 3.25 Ã— 10â€“5, and p = 0.014, respectively). Each p-value reflects comparisons of mutant to its respective WT. The symbol â€œ*â€ denotes statistical significance. For statistical analyses, see section â€œMaterials and Methods.â€ We harvested oocytes from two frogs to perform these experiments.
	FIGURE 7. Normo-osmotic swelling of AQP7 and GlpF (Â± AQP1) expressing oocytes. (Aâ€“C) Oocyte swelling rates for AQP7 mutant channels expressed alone or co-expressed with AQP1 as [glycerol]o in increased in a normo-osmotic buffer. (Dâ€“F) Oocyte swelling rates for GlpF mutant channels expressed alone or co-expressed with AQP1 as [glycerol]o in increased in a normo-osmotic buffer. All data points represent mean Â± S. E. M. These data and the number of oocytes and oocyte preparations/cRNA injections used to generate each point are detailed in Supplementary Tables S3, S4.

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