Aquaglyceroporins, one channel for two molecules (original) (raw)
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Proceedings of the National Academy of Sciences, 2010
Aquaporins are homotetrameric channel proteins, which allow the diffusion of water and small solutes across biological membranes. According to their transport function, aquaporins can be divided into "orthodox aquaporins", which allow the flux of water molecules only, and "aquaglyceroporins", which facilitate the diffusion of glycerol and other small solutes in addition to water. The contribution of individual residues in the pore to the selectivity of orthodox aquaporins and aquaglyceroporins is not yet fully understood. To gain insights into aquaporin selectivity, we focused on the sequence variation and electrostatics of their channels. The continuum Poisson-Boltzmann electrostatic potential along the channel was calculated and compared for ten three-dimensionalstructures which are representatives of different aquaporin subfamilies, and a panel of functionally characterized mutants, for which high-accuracy three-dimensional-models could be derived. Interestingly, specific electrostatic profiles associated with the main selectivity to water or glycerol could be identified. In particular: (i) orthodox aquaporins showed a distinctive electrostatic potential maximum at the periplasmic side of the channel around the aromatic/Arg (ar/R) constriction site; (ii) aquaporin-0 (AQP0), a mammalian aquaporin with considerably low water permeability, had an additional deep minimum at the cytoplasmic side; (iii) aquaglyceroporins showed a rather flat potential all along the channel; and (iv) the bifunctional protozoan PfAQP had an unusual all negative profile. Evaluation of electrostatics of the mutants, along with a thorough sequence analysis of the aquaporin pore-lining residues, illuminated the contribution of specific residues to the electrostatics of the channels and possibly to their selectivity. channel proteins | electrostatic potentials | pore analysis | comparative analysis
Glycerol facilitator GlpF and the associated aquaporin family of channels
Current Opinion in Structural Biology, 2003
The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.
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.
Members of the superfamily of major intrinsic proteins (MIPs) facilitate water and solute permeability across cell membranes and are found in sources ranging from bacteria to humans. Aquaporin and aquaglyceroporin channels are the prominent members of the MIP superfamily. Experimental studies show that MIPs are involved in important phys- iological processes in mammals and plants. They are implicated in several human diseases and are considered to be attractive drug targets for a wide range of diseases such as cancer, brain edema, epilepsy, glaucoma, and congestive heart failure. Three-dimensional structures of MIP channels from diverse sources reveal that MIPs adopt a unique conserved hourglass helical fold consisting of six transmembrane heli- ces (TM1–TM6) and two half-helices (LB and LE). Conserved NPA motifs near the center and the aromatic/arginine selectivity filter (Ar/R SF) toward the extracellular side consti- tute two narrow constriction regions within the channel. Structural knowledge com- bined with simulation studies have helped to investigate the role of these two constriction regions in the transport and selectivity of the solutes. With the availability of many genome sequences from diverse species, a large number of MIP genes have been identified. Homology models of 1500 MIP channels have been used to derive structure-based sequence alignment of TM1–TM6 helices and the two half-helices LB and LE. Thirteen residues are highly conserved in different transmembrane helices and half-helices. High group conservation of small and weakly polar residues is observed in 27 positions at the interface of two interacting helices. Thus, although the MIP sequences are diverse, the hourglass helical fold is maintained during evolution with the conservation of these 40 positions within the transmembrane region. We have pro- posed a generic structure-based numbering scheme for the MIP channels that will facil- itate easier comparison of the MIP sequences. Analysis of Ar/R SF in all 1500 MIPs indicates the extent of diversity in the four residues that form this narrow region. Certain residues are completely avoided in the SF, even if they have the same chemical nature as that of the most frequently observed residues. For example, arginine is the most pre- ferred residue in a specific position of Ar/R SF, whereas lysine is almost always avoided in any of the four positions. MIP channels with highly hydrophobic or hydrophilic Ar/R SF have been identified. Similarly, there are examples of MIP channels in which all four res- idues of Ar/R SF are bulky, thus almost occluding the pore. Many plant MIPs possess small residues at all SF positions, resulting in a larger pore diameter. A majority of MIP channels are yet to be functionally characterized, and their in vivo substrates are not yet identified. A complete understanding of the relationship between the nature of Ar/R SF and the solutes that are transported is required to exploit MIP channels as potential drug targets.
Oligomerization of water and solute channels of the major intrinsic protein (MIP) family
Kidney International, 2001
(250 to 290 amino acids), and the high conservation intrinsic protein (MIP) family. Water and small solute fluxes throughout the MIP family may indicate a common fold: through cell membranes are ensured in many tissues by seleca NH 2 cytosolic portion followed by a hydrophobic stretch tive pores that belong to the major intrinsic protein family of six transmembrane helices ). Among highly (MIP). This family includes the water channels or aquaporins conserved amino acids in the family, two repetitions of (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of Asp, Pro, Ala residues (NPA box) localized in the B and representatives of each subfamily. Following solubilization in E loops draw the sequence signature of the family (Fig. the nondenaturing detergents n-octyl-glucoside (OG) and Tri-1B). The folding of these two loops in the membrane ton X-100 (T-X100), AQPs remain in their native homotetrabilayer is proposed to be responsible of the pore formameric state, while GlpF always behaves as a monomer. Solute facilitators are fully solubilized by the detergent N-lauroyl sartion and solute movement across the membrane [2, 3]. cosine (NLS), while AQPs are not. Analyses of mutants and Interestingly, AQPs are widely distributed in bacteria, chimeras demonstrate a close correlation between the water plants, and animals, while GlpFs have been characterized transport function and the resistance to NLS solubilization. only within microorganisms such as bacteria or yeast. In Thus, AQPs and solute facilitators exhibit different behaviors mammals, ten MIPs have been cloned and functionally in mild detergents; this could reflect differences in quaternary organization within the membranes. We propose that the oligocharacterized. Some of them, such as AQP1, AQP3, and merization state or the strength of self-association is part of the AQP4, are widely distributed in the body [4]. In contrast, mechanisms used by MIP proteins to ensure solute selectivity.
Structure of a Glycerol-Conducting Channel and the Basis for Its Selectivity
Science, 2000
Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane. We report the crystal structure of the Escherichia coli glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution. Glycerol molecules line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms. Two conserved aspartic acid-proline-alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded.
Control of the Selectivity of the Aquaporin Water Channel Family by Global Orientational Tuning
Science, 2002
Aquaporins are transmembrane channels found in cell membranes of all life forms. We examine their apparently paradoxical property, facilitation of efficient permeation of water while excluding protons, which is of critical importance to preserving the electrochemical potential across the cell membrane. We have determined the structure of the Escherichia coli aquaglyceroporin GlpF with bound water, in native (2.7 angstroms) and in W48F/F200T mutant (2.1 angstroms) forms, and carried out 12-nanosecond molecular dynamics simulations that define the spatial and temporal probability distribution and orientation of a single file of seven to nine water molecules inside the channel. Two conserved asparagines force a central water molecule to serve strictly as a hydrogen bond donor to its neighboring water molecules. Assisted by the electrostatic potential generated by two half-membrane spanning loops, this dictates opposite orientations of water molecules in the two halves of the channel, a...
Water and glycerol permeation through the glycerol channel GlpF and the aquaporin family
Journal of Synchrotron Radiation, 2004
The 2.2 Å resolution crystal structure of GlpF, an E.coli aquaporin that facilitates the flow of glycerol, water and other small solutes, provides much insight into the molecular function and selectivity of aquaporins. Using GlpF and its atomic structure as a paradigm for the ten highly conserved human aquaporins, site-directed mutagenesis has been used to mutate residues that are possibly integral to the structure and function of different aquaporins. X-ray crystallography and other biophysical and molecular simulation methods allows for assessment of these changes at the structural and functional level. Initial attempts to convert the glycerol specific properties of GlpF towards a water specific aquaporin resulted in the shifting of GlpF channel properties towards that of the water aquaporins. This result reveals the great possibility of emulating and deciphering the function of other aquaporins with GlpF via mutagenesis and investigation of structure and function.
Selectivity and conductance among the glycerol and water conducting aquaporin family of channels
FEBS Letters, 2003
The atomic structures of a transmembrane water plus glycerol conducting channel (GlpF), and now of aquaporin Z (AqpZ) from the same species, Escherichia coli, bring the total to three atomic resolution structures in the aquaporin (AQP) family. Members of the AQP family each assemble as tetramers of four channels. Common helical axes support a wider channel in the glycerol plus water channel paradigm, GlpF. Water molecules form a single hydrogen bonded ¢le throughout the 28 A î long channel in AqpZ. The basis for absolute exclusion of proton or hydronium ion conductance through the line of water is explored using simulations. (R.M. Stroud).
The mechanism of glycerol conduction in aquaglyceroporins
2001
to be ion conducting at low pH [3]. Two structures of human aquaporin 1 (AQP1) determined by electron crys-405 N. Mathews Urbana, Illinois 61801 tallography were previously reported [4, 5]. The structures are atomic models derived from 2D crystallogra-2 Department of Chemistry Technical University of Denmark phy data at 3.8 Å and 3.7 Å in plane and 4.4 Å and 6.0 Å normal resolution, respectively, and both identify DTU 207 DK-2800 Lyngby AQP1 as a homotetrameric membrane channel [4, 5]. The X-ray structure of the Escherichia coli glycerol facili-Denmark tator, GlpF, a homotetramer highly similar to AQP1, was reported at a resolution of 2.2 Å [6]. The GlpF structure included the positions of three glycerol molecules and Summary two water molecules per monomer, confirming the GlpF selectivity for both substrates. GlpF also stereo-and Background: The E. coli glycerol facilitator, GlpF, seenantio-selectively conducts other polyalcohols (aldilectively conducts glycerol and water, excluding ions tols) [6, 7]. Water and glycerol influxes in reconstituted and charged solutes. The detailed mechanism of the liposomes are increased by GlpF 10-fold and 100-to glycerol conduction and its relationship to the character-1000-fold, respectively, relative to the pure liposome [2, istic secondary structure of aquaporins and to the NPA 6-8]. In terms of water permeability, GlpF is found to be motifs in the center of the channel are unknown. about one order of magnitude less efficient than AqpZ, the E. coli variant of human AQP1 [2, 8].