Structural clues in the sequences of the aquaporins (original) (raw)
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Journal of Biological Chemistry, 2002
We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the Cterminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.
The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold
Small
Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence-and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of 4 representatives, we identify key aspects of AQP stability, gating, selectivity, pore geometry and oligomerization, with a potential impact on channel functionality. We challenge the general view of AQPs possessing a continuous open water pore and depict that AQPs selectivity is not exclusively shaped by pore lining residues but also by the relative arrangement of transmembrane helices. Moreover, our analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, we establish a novel numbering scheme of the conserved AQP scaffold facilitating direct comparison and prediction of potential structural effects of e.g. disease-causing mutations. Additionally, our results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications. 1. Introduction Aquaporins (AQPs), part of a larger family of major intrinsic proteins, are one of the best studied protein families with currently 20 non-redundant high-resolution structures (≤3,70 Å) solved. Since their first discovery in 1992 by Peter Agre and coworkers (1, 2), thirteen different types of aquaporins (AQP0-12) were discovered in mammals (3). The narrow AQP pores combine enormous permeability, conducting water in a single-file manner close to the diffusion limit of water in bulk, with exceptional selectivity (4). A subset of AQPs, the aquaglyceroporins (AQGPs), paralogs of AQPs, are also able to conduct glycerol and other small neutral solutes (5, 6). Bacteria also express members of AQPs and AQGPs, generally functioning with one copy of each paralog and, interestingly, some lacking both. Unicellular eukaryotes and fungi follow a similar pattern, with a clear division between AQPs or AQGPs and a heterogeneous distribution in the number of copies of each paralog in the different genera (7). So far, no archaea has been found that possesses both paralogs concurrently. Plants exhibit the highest AQP diversity, with five main subfamilies (plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs)), which are each further divided into subgroups (7). Furthermore, in primitive plant species, two additional subfamilies, GLPF-like intrinsic proteins (GIPs) and hybrid intrinsic proteins (HIPs), have been found (8). However, the full diversity of AQ(G)Ps is still not represented by the numerous high-resolution structures, as exemplified by only three plant aquaporin structures and none of the unorthodox AQ(G)Ps (represented by AQP11 and 12 in mammals).
Molecular Aspects of Medicine, 2012
After a decade of work on the water permeability of red blood cells (RBC) Benga group in Cluj-Napoca, Romania, discovered in 1985 the first water channel protein in the RBC membrane. The discovery was reported in publications in 1986 and reviewed in subsequent years. The same protein was purified by chance by Agre group in Baltimore, USA, in 1988, who called in 1991 the protein CHIP28 (CHannel forming Integral membrane Protein of 28 kDa), suggesting that it may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 the Agre group identified CHIP28's water transport property. One year later CHIP28 was named aquaporin 1, abbreviated as AQP1. In this review the molecular structure-function relationships of AQP1 are presented. In the natural or model membranes AQP1 is in the form of a homotetramer, however, each monomer has an independent water channel (pore). The three-dimensional structure of AQP1 is described, with a detailed description of the channel (pore), the molecular mechanisms of permeation through the channel of water molecules and exclusion of protons. The permeability of the pore to gases (CO 2 , NH 3 , NO, O 2) and ions is also mentioned. I have also reviewed the functional roles and medical implications of AQP1 expressed in various organs and cells (microvascular endothelial cells, kidney, central nervous system, eye, lacrimal and salivary glands, respiratory apparatus, gastrointestinal tract, hepatobiliary compartments, female and male reproductive system, inner ear, skin). The role of AQP1 in cell migration and angiogenesis in relation with cancer, the genetics of AQP1 and mutations in human subjects are also mentioned. The role of AQP1 in red blood cells is discussed based on our comparative studies of water permeability in over 30 species.
Molecular Aspects of Medicine, 2012
The importance of discovery of the water channel proteins (WCPs) for biology has been very well emphasized by Wolburg et al. (2011): ''The detection of water-specific membrane channels in red blood cells belongs to the fundamental discoveries in biology of the twentieth century (Benga et al., 1986a; Denker et al., 1988; Preston et al., 1992).'' In the over 25 years that passed since the first discovery in the red blood cell membrane of the first water channel protein (Benga et al., 1986a,b) this domain of science became a very hot area of research in molecular cell biology, biochemistry and biophysics, with increasing physiological and medical implications. As thousands of publications appeared on these topics there is a need for overviews of structure-function relationships and medical implications of water channel proteins (WCPs) at the molecular level. This Special Issue of ''Molecular Aspects of Medicine'' has this aim. An important aspect is to clarify the nomenclature of WCPs, taking into account that various names used by different authors for the same proteins, or classify the same proteins in different classes. Consequently, in this issue the definition and nomenclature of WCPs, members is presented (Benga, 2012). WCPs, a family of proteins, belonging to the Membrane Intrinsic Proteins (MIPs) superfamily with more than 1000 members, are characterized by the homotetrameric structure with each monomer having a specific three-dimensional structure with a pore that provides a pathway for water permeation. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the ''signature'' sequence of WCPs, a family of proteins including three subfamilies. (1) Aquaporins (abbreviated as AQPs) are mainly water selective or specific water channels, also named by various authors as ''orthodox'', ''ordinary'', ''conventional'', ''classical'', ''pure'', ''normal'', or ''sensu strictu'' aquaporins; (2) Aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named ''superaquaporins'', ''aquaporins with unusual (or deviated) NPA boxes'', ''subcellular aquaporins'', or ''sip-like aquaporins''. So far, 13 WCPs have been discovered in mammals. Most authors agree that seven of these are aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5. AQP6, and AQP8), four are aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10), whereas AQP11 and AQP12 are S-aquaporins.
Structure and Function of Aquaporins: the Membrane Water Channel Proteins
Biointerface Research in Applied Chemistry, 2021
Aquaporins are integral membrane proteins which are also known as water channel proteins. They aid quick transportation of water across membranes and are important in controlling cell volume and transcellular water passage. Aquaporins are present in organisms, and they vary from archaea and bacteria to plants and animals. They are also found in insects and yeast. Presently, 13 mammalian aquaporins (AQP0 to AQP12) have been cloned and identified in every tissue in the body. These aquaporins are alike in basic structure with monomers containing six transmembrane and two short helical segments that enclose cytoplasmic and extracellular vestibules linked by aqueous pore. They have distinctive structures that define their functions, mode of action, and even their various control methods. Phylogenetic analysis of aquaporin consists of aquaporins, glycerol facilitators, plasma membrane integral proteins of plants, tonoplast integral proteins of plants, nodules of plants, and AQP8s. Aquapor...
The channel architecture of aquaporin 0 at a 2.2-A resolution
Proceedings of the National Academy of Sciences, 2004
We determined the x-ray structure of bovine aquaporin 0 (AQP0) to a resolution of 2.2 Å. The structure of this eukaryotic, integral membrane protein suggests that the selectivity of AQP0 for water transport is based on the identity and location of signature amino acid residues that are hallmarks of the water-selective arm of the AQP family of proteins. Furthermore, the channel lumen is narrowed only by two, quasi-2-fold related tyrosine side chains that might account for reduced water conductance relative to other AQPs. The channel is functionally open to the passage of water because there are eight discreet water molecules within the channel. Comparison of this structure with the recent electron-diffraction structure of the junctional form of sheep AQP0 at pH 6.0 that was interpreted as closed shows no global change in the structure of AQP0 and only small changes in side-chain positions. We observed no structural change to the channel or the molecule as a whole at pH 10, which could be interpreted as the postulated pH-gating mechanism of AQP0-mediated water transport at pH >6.5. Contrary to the electron-diffraction structure, the comparison shows no evidence of channel gating induced by association of the extracellular domains of AQP0 at pH 6.0. Our structure aids the analysis of the interaction of the extracellular domains and the possibility of a cell-cell adhesion role for AQP0. In addition, our structure illustrates the basis for formation of certain types of cataracts that are the result of mutations.
NPA motifs play a key role in plasma membrane targeting of aquaporin-4
IUBMB Life, 2010
The two highly conserved NPA motifs (asparagine-prolinealanine, NPA) are the most important structural domains that play a crucial role in water-selective permeation in aquaporin water channels. However, the functions of NPA motifs in aquaporin (AQP) biogenesis remain largely unknown. Few AQP members with variations in NPA motifs such as AQP11 and AQP12 do not express in the plasma membrane, suggesting an important role of NPA motifs in AQP plasma membrane targeting. In this study, we examined the role of the two NPA motifs in AQP4 plasma membrane targeting by mutagenesis. We constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in AQP4-M1 and AQP4-M23 isoforms and analyzed their expression patterns in transiently transfected FRT and COS-7 cells. Western blot analysis showed similar protein bands of all the AQP4 mutants and the wild-type AQP4. AQP4 immunofluorescence indicated that deletion of one or both NPA motifs resulted in defective plasma membrane targeting, with apparent retention in endoplasmic reticulum (ER). The A99T mutant mimicking AQP12 results in ER retention, whereas the A99C mutant mimicking AQP11 expresses normally in plasma membrane. Furthermore, the AQP4-M1 but not the M23 isoform with P98A substitution in the first NPA motif can target to the plasma membrane, indicating an interaction of Nterminal sequence of AQP4-M1 with the first NPA motif. These results suggest that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs may interact with other structural domains in the regulation of membrane trafficking during aquaporin biogenesis. 2010 IUBMB IUBMB Life, 62(3): 222-226, 2010
A refined structure of human aquaporin-1
FEBS Letters, 2001
A re ned structure of the human water channel aquaporin 1 AQP1 is presented. The model rests on a the high resolution x-ray structure of the homologous bacterial glycerol transporter GlpF, b electron-crystallographic data of AQP1 at 3.8 A resolution and c a multiple sequence alignment of the aquaporin superfamily. The computed crystallographic R and free R values 36.7 and 37.8 for the re ned structure are signi cantly lower in comparison to two previous AQP1 models. Together with improved geometrical normality scores and an enhanced stability in molecular dynamics simulations, this implicates a signi cant improvement of the AQP1 structure. Comparisons with the previous models of the AQP1 structure show signi cant di erences, not only in the loop regions where the experimental densities are relatively weak, but also in the transmembrane region facing the core of the water channel. Implications of these di erences for the structural integrity of the protein as well as for the water permeability are discussed.
The 3.7 Å projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer
EMBO reports, 2000
GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of ∼80 Å side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 Å resolution. Cryoelectron microscopy provided a 3.7 Å projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.
The Three-Dimensional Structure of Aquaporin-1
Nature, 1997
letters to nature 624 NATURE | VOL 387 | 5 JUNE 1997 with RsaI before cDNA subtraction. cDNA prepared from RNA isolated on day 3 of induction was subtracted using cDNA prepared from undifferentiated cell RNA using a polymerase chain reaction (PCR)-select cDNA subtraction kit (Clonetech). The remaining cDNAs were randomly subcloned into a T-vector (Promega). Sixty-six clones were sequenced, and their sequences were compared with those in the GenBank/EMBL/DDBJ database. One clone (5m-1) was found to encode the 3Ј-UTR of a previously isolated seven-span orphan receptor 9 . The cDNA corresponding to the ORF of the orphan receptor was amplified by PCR from 1 g of human genomic DNA. The primers used for PCR were 5Ј-CGGGATCCCGATGGCGTCAGGAAACCCTTG-3Ј (sense), and 5Ј-GGAATTCCTAGTTCAGTTCGTTTAACTTG-3Ј (antisense). The PCR conditions were as follows: denaturation at 96 ЊC for 1 min, annealing at 55 ЊC for 1 min, elongation at 72 ЊC for 3 min; 30 cycles. The amplified fragment was randomly labelled with [ 32 P]dCTP, and was used to screen an HL-60 cDNA library, which was constructed in Zap-II (Stratagene) from 5 g poly(A) þ RNA of HL-60 cells differentiated by exposure to 1 M retinoic acid for 3 days. 5 ϫ 10 5 independent clones were screened and five clones (HL-1 to HL-5) were isolated by high-stringency washing. DNA sequencing revealed that HL-1 and HL-5 contain identical full-length ORFs. The ORF of HL-5 was subcloned in the mammalian expression vector pcDNA3 (Invitrogen), and the resulting plasmid designated pLTBR. Northern blot analysis. Poly(A) þ RNA (3 g) from HL-60 and U-937 cells was electrophoresed in a 1% agarose gel, and transferred to a Hybond-N nylon membrane (Amersham). Human multiple tissue northern blot filters I and II were purchased from Clonetech. The filters were hybridized with [ 32 P]dCTPlabelled ORF of the HL-5 clone or a human glutaraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA (Clonetech) in hybridization buffer containing 4 ϫ SSC, 5 ϫ Denhardt 0 s solution, 0.2% SDS, 200 g ml Ϫ 1 salmon sperm DNA, 50% formamide at 42 ЊC for 24 h. The filters were washed in 0:1 ϫ SSC, 0.1% SDS at 65 ЊC and subjected to autoradiography. Expression in mammalian cells and characterization. Cos-7, HEK-293 and C6-15 cells were cultured in DMEM, and CHO cells in F12 medium. Both media contained 10% fetal calf serum. DNA transfection was performed by lipofection using Transfectam (Gibco BRL) for Cos-7, HEK-293, and CHO cells 28 , or a calcium phosphate method for C6-15 glioma cells 27 . The membrane fractions were prepared as described 14 . Stable transformants were selected with 1 g l −1 Geneticin (Gibco BRL), and cloned by limiting dilution. Clones that showed increases in intracellular calcium following exposure to 100 nM LTB 4 were selected for further analysis. LTB 4 binding assay and measurements of cAMP, InsP 3 , and intracellular calcium were carried out using established protocols 19,28 . Chemotaxis assay. Polycarbonate filters with 8-m pores (Neuroprobe) were coated with 13.3 g ml Ϫ 1 fibronectin (Sigma) in PBS for 60 min 29 . A dry coated filter was placed on a 96-blind well chamber (Neuroprobe) containing the indicated amounts of LTB 4 , and the CHO cells (200 l, 8 ϫ 10 4 per well) were added to the top wells. The ligand solution and cell suspension were prepared in the same buffer (F-12 medium containing 0.1% BSA). After incubation at 37 ЊC in 5% CO 2 for 4 h, the filter was disassembled. The cells on the filter were fixed with methanol and stained with a Diff-Quick staining kit (International Reagents Corp.). The upper side of the filter was then scraped free of cells. The number of cells that migrated to the lower side was determined by measuring optical densities at 595 nm using a 96-well microplate reader Model 3550 (Biorad).