The 4.5Å Structure of Human AQP2

Schenk, Andreas; Werten, Paul J.L.; Scheuring, Simon; Groot, Bert L. de; Müller, Shirley A.; Stahlberg, Henning; Philippsen, Ansgar; Engel, Andréas

doi:10.1016/j.jmb.2005.04.030

Cited by 71 publications

(39 citation statements)

References 62 publications

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“…Human AQP2 expressed in insect cells (17) has previously yielded 2D crystals diffracting to 4.5 Å resolution (18). Extensive efforts to produce better diffracting 3D crystals using full-length human AQP2 produced in insect cells failed to sufficiently improve the diffraction.…”

Section: Resultsmentioning

confidence: 99%

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

Frick

Eriksson

Mattia

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

111

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Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd 2+ -ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.membrane protein | X-ray crystallography | water channel protein W ater is the major ingredient of the human body, constituting 55-65% of our total body weight (1). Water homeostasis is maintained by the kidneys, which filter ∼180 L of primary urine every day. Although most water is constitutively reabsorbed in the proximal tubules and descending limbs of Henle (2), the body's water balance is fine-tuned by regulated water reabsorption, which takes place in the kidney collecting duct. Water reabsorption is mediated by aquaporins, membranebound water channels, of which seven of the 13 human isoforms have been located in the human kidney (3). Of these, human aquaporin 2 (AQP2) is present in the principal cells of the collecting duct and is responsible for regulated water reabsorption.AQP2 is stored in intracellular vesicles under water-saturating conditions. When the levels of the pituitary antidiuretic hormone arginine vasopressin (AVP) are elevated in response to dehydration or hypernatremia, AVP binding to the vasopressin 2 receptor (V2R) in the basolateral membrane stimulates an increase in intracellular cAMP. This triggers the phosphorylation of Ser256 in the AQP2 C terminus by protein kinase A (PKA) and flags the protein for trafficking from storage vesicles to the apical membrane (4-6). AVP also triggers additional phosphorylation at Ser264 and Ser269 (7,8), with all three sites being phosphorylated in AQP2s targeted to the plasma membrane (9). The resulting redistribution of AQP2 increases transcellular water permeability and concentrates urine (Fig. S1). Once correct water balance is restored, AQP2 is internalized through ubiquitinmediated endocytosis and redirected to storage vesicles or targeted for degradation (10-12).Because of its central role in water homeostasis, dysregula...

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Section: Resultsmentioning

confidence: 99%

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

Frick

Eriksson

Mattia

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

111

View full text Add to dashboard Cite

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“…The mass-per-length is a useful if not essential parameter defining the structure of filaments [25], indicating the number of strands present [26][27][28][29] and in the case of helices restricting the number of possible assembly rules [30][31][32]. Similarly, the massper-area defines the unit cell stoichiometry in 2D crystals or the number of layers in a sheet-like structure [33][34][35][36], information that is otherwise only accessible by chance at their edges, or by atomic force microscopy [36] [37]. Further, in conjunction with a lipid assay by analytical ultracentrifugation mass-per-area measurements can be used to confirm the predicted packing of membrane proteins in two-dimensional crystalline arrays [35].…”

Section: Introductionmentioning

confidence: 99%

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Müller¹,

Engel²

2006

Chimia

Self Cite

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scanning transmission electron microscopes can both measure the mass of single protein complexes and take amazingly clear images thereof, offering a wide range of applications in structural biology. The principle of mass measurement is presented and discussed and the scope of scanning transmission electron microscopy is illustrated by selected examples. Keywords:Mass determination · scanning transmission electron microscopy · sTEM unstained proteins to be clearly visualised under low dose conditions. Each pixel of the digital image is a quantitative scattering experiment from which the mass of the irradiated volume can be calculated [9]. Thus, the mass of a single protein complex can be determined by integrating over all pixel values that lie within its boundary. The method is applicable to macromolecules of widely varying mass and form (reviewed in [10]). When combined with SDS-gel electrophoresis and, where available, sequence information, STEM mass measurements serve to define protein stoichiometry. Further, as electron energy loss spectroscopy [11][12] or energy dispersive X-ray spectroscopy [13] can be performed while imaging, the STEM also offers the possibility of determining the distribution of specific elements in protein complexes. Indeed the feasibility of mapping single atoms bound to protein molecules has recently been demonstrated [14].The high contrast delivered by the dark-field detector system [15] can also be exploited in negative or positive stain microscopy [16]. Here the STEM yields clear single-shot images that are free from phase contrast fringes. These images frequently provide information only otherwise visible after averaging several hundred projections recorded with a transmission electron microscope (TEM). They thus make it possible to document the presence of distinct protein conformations, information that would be lost by averaging techniques. Indeed, STEM images of both negatively stained [17] and unstained [18] protein complexes have been used to reconstruct their 3D structure.The few dedicated STEMs employed in biology today are invaluable. In particular, the coupling of image and mass gives them the power to answer questions not resolvable by ultracentrifugation or mass spectrometry. Here we discuss the use of the STEM both as an imaging tool and to measure mass, and examine the uncertainties to be expected in STEM mass data sets. The Importance and Versatility of STEM Mass MeasurementsWorldwide, only four laboratories routinely use dedicated STEMs to measure the mass of biological samples. Their facilities are open for external collaborations and the many papers in the literature containing STEM mass measurements document the importance of this rare technique to the scientific community. Although the precision of STEM cannot match that of the mass spectrometer, its capability to measure an enormously wide mass range, its independence from shape assumptions and the presence of an image make the STEM an invaluable tool.The methodology of mass spectrometry has develop...

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“…X-ray and electron diffraction studies have yielded crystal structures of mammalian AQP0 (4-6), AQP1 (7,8), AQP2 (9), and AQP4 (10), plant SoPIP2;1 (11,12), bacterial AQPZ (13,14), and GlpF (15), and the archaeal AQPM (16). These structures establish that phylogenetically and functionally diverse AQPs arrange as homotetramers, each protomer containing six highly conserved transmembrane (TM) ␣-helices.…”

mentioning

confidence: 96%

High-resolution x-ray structure of human aquaporin 5

Horsefield

Nordén²,

Fellert³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

204

180

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Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-Å resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.membrane protein ͉ trafficking ͉ crystallography ͉ water channel protein ͉ heterologous overexpression A quaporins (1) facilitate the flow of water across cellular membranes while preserving ion concentration gradients. By maintaining water homeostasis within cells, aquaporin family members play essential physiological roles within all kingdoms of life. They form a large superfamily containing both pure water channels, and channels also permeable to other small polar molecules such as glycerol (2). Thirteen human aquaporin (AQP) isoforms have been identified, and they govern a broad spectrum of physiological functions (2, 3). Examples include concentration of urine in the kidneys, release of tears and maintenance of lens transparency in the eye, maintenance of water homeostasis within the brain, the extrusion of sweat from the skin, control of glycerol concentration in fat metabolism, and facilitation of cell migration during angiogenesis.X-ray and electron diffraction studies have yielded crystal structures of mammalian AQP0 (4-6), AQP1 (7, 8), AQP2 (9), and AQP4 (10), plant SoPIP2;1 (11, 12), bacterial AQPZ (13,14), and GlpF (15), and the archaeal AQPM (16). These structures establish that phylogenetically and functionally diverse AQPs arrange as homotetramers, each protomer containing six highly conserved transmembrane (TM) ␣-helices. Two half-helices form a pseudoseventh TM helix because of the insertion of loops B and E into the membrane from opposite sides, placing both copies of the highly conserved Asn-Pro-Ala (NPA) AQP signature motif near the center of the water channel. The conserved aromatic/arginine (ar/R) constriction region imposes substrate selectivity on the channel (17). A consensus has emerged from molecular dynamics simulations regarding the mechanism of water transport and ion exclusion (18) establishing that an electrostatic potential barrier peaking at the NPA region prevents the cotranslocation of protons through the channel.Although the tissue-specific expressio...

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The 4.5Å Structure of Human AQP2

Cited by 71 publications

References 62 publications

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

High-resolution x-ray structure of human aquaporin 5

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