The 6.9-A structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli

The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy. The 6.9-A density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel. While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction. Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel. Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side. This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity.     

Diffusion of glycerol through Escherichia coli aquaglyceroporin GlpF

The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 μs. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape.     

The mechanism of glycerol conduction in aquaglyceroporins.

BACKGROUND: The E. coli glycerol facilitator, GlpF, selectively conducts glycerol and water, excluding ions and charged solutes. The detailed mechanism of the glycerol conduction and its relationship to the characteristic secondary structure of aquaporins and to the NPA motifs in the center of the channel are unknown. RESULTS: Molecular dynamics simulations of GlpF reveal spontaneous glycerol and water conduction driven, on a nanosecond timescale, by thermal fluctuations. The bidirectional conduction, guided and facilitated by the secondary structure, is characterized by breakage and formation of hydrogen bonds for which water and glycerol compete. The conduction involves only very minor changes in the protein structure, and cooperativity between the GlpF monomers is not evident. The two conserved NPA motifs are strictly linked together by several stable hydrogen bonds and their asparagine side chains form hydrogen bonds with the substrates passing the channel in single file. CONCLUSIONS: A complete conduction of glycerol through the GlpF was deduced from molecular dynamics simulations, and key residues facilitating the conduction were identified. The nonhelical parts of the two half-membrane-spanning segments expose carbonyl groups towards the channel interior, establishing a curve-linear pathway. The conformational stability of the NPA motifs is important in the conduction and critical for selectivity. Water and glycerol compete in a random manner for hydrogen bonding sites in the protein, and their translocations in single file are correlated. The suggested conduction mechanism should apply to the whole family.     

The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer

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.     

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p.

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.     

Glycerol conductance and physical asymmetry of the Escherichia coli glycerol facilitator GlpF

The aquaglyceroporin GlpF is a transmembrane channel of Escherichia coli that facilitates the uptake of glycerol by the cell. Its high glycerol uptake rate is crucial for the cell to survive in very low glycerol concentrations. Although GlpF allows both influx and outflux of glycerol, its structure, similar to the structure of maltoporin, exhibits a significant degree of asymmetry. The potential of mean force characterizing glycerol in the channel shows a corresponding asymmetry with an attractive vestibule only at the periplasmic side. In this study, we analyze the potential of mean force, showing that a simplified six-step model captures the kinetics and yields a glycerol conduction rate that agrees well with observation. The vestibule improves the conduction rate by 40% and 75% at 10- micro M and 10-mM periplasmic glycerol concentrations, respectively. In addition, neither the conduction rate nor the conduction probability for a single glycerol (efficiency) depends on the orientation of GlpF. GlpF appears to conduct equally well in both directions under physiological conditions.     

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.     

Proton exclusion by an aquaglyceroprotein: a voltage clamp study

BACKGROUND INFORMATION: In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. RESULTS: To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7x10(-14) cm(3).subunit(-1).s(-1). Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H(+) (OH(-)) to H(2)O molecules transport stoichiometry of 2x10(-9). A significant GlpF-mediated ion conductivity was also not detectable. CONCLUSIONS: The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.     

Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression

The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh. HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue. The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46. But in neither ccpA nor hprK mutants was expression of a glpF'-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF'-lacZ fusion and to partial relief from CCR. CCR completely disappeared in DeltatglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK-requiring CCR mechanism seems to be based on insufficient synthesis of glycerol-3-P, as CCR of glpFK was absent in ccpA mutants growing on glycerol-3-P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P-His-HPr. P-GlpK is much more active than GlpK, and the absence of P~GlpK formation in DeltaptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol-3-P will be formed in glycerol and glucose-exposed cells (inducer exclusion). The uptake of glycerol-3-P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF'-lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P-GlpK.     

What makes an aquaporin a glycerol channel? A comparative study of AqpZ and GlpF

The recent availability of high-resolution structures of two structurally highly homologous, but functionally distinct aquaporins from the same species, namely Escherichia coli AqpZ, a pure water channel, and GlpF, a glycerol channel, presents a unique opportunity to understand the mechanism of substrate selectivity in these channels. Comparison of the free energy profile of glycerol conduction through AqpZ and GlpF reveals a much larger barrier in AqpZ (22.8 kcal/mol) than in GlpF (7.3 kcal/mol). In either channel, the highest barrier is located at the selectivity filter. Analysis of substrate-protein interactions suggests that steric restriction of AqpZ is the main contribution to this large barrier. Another important difference is the presence of a deep energy well at the periplasmic vestibule of GlpF, which was not found in AqpZ. The latter difference can be attributed to the more pronounced structural asymmetry of GlpF, which may play a role in attracting glycerol.     

A water channel of the nematode C.elegans and its implications for channel selectivity of MIP proteins

A genome project focusingon the nematode Caenorhabditis elegans has demonstrated thepresence of eight cDNAs belonging to the major intrinsic proteinsuperfamily. We functionally characterized one of these cDNAs namedC01G6.1. Injection of C01G6.1 cRNA increased the osmotic waterpermeability (P_f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed toincrease the glycerol permeability. It has been speculated that the MIPfamily may be separated into two large subfamilies based on thepresence or absence of two segments of extra amino acid residues (~15amino acids) at the second and third extracellular loops. BecauseC01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with thoseof AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that ofwild-type AQP-CE1, although the values of P_f andurea permeability were decreased by 39-74% and 28-65%,respectively. These results suggest that the two segments of extraamino acid residues may not contribute to channel selectivity orformation of the route for small solutes.

     

The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function

The vestibule loop regions of aquaglyceroporins are involved in accumulation of glycerol inside the channel pore. Even though most loop regions do not show high sequence similarity among aquaglyceroporins, loop E is highly conserved in aquaglyceroporins, but not in members of the homologous aquaporins. Specifically, a tryptophan residue is extremely conserved within this loop. We have investigated the role of this residue (Trp219) that deeply protrudes into the protein and potentially interacts with adjacent loops, using the E. coli aqualgyeroporin GlpF as a model. Replacement of Trp219 affects the activity of GlpF and impairs the stability of the tetrameric protein. Furthermore, we have identified an amino acid cluster involving Trp219 that stabilizes the GlpF tetramer. Based on our results we propose that Trp219 is key for formation of a defined vestibule structure, which is crucial for glycerol accumulation as well as for the stability of the active GlpF tetramer.     

DIP: a member of the MIP family of membrane proteins that is expressed in mature seeds and dark-grown seedlings of Antirrhinum majus

DiP , a gene from Antirrhinum majus , which encodes a protein with striking homology to other integral membrane proteins, was cloned. The gene was specifically expressed in mature seeds and during seedling germination, particularly in cotyledons of seedlings grown in the dark. The deduced product, called DiP, for dark intrinsic protein, shows strong homology with the MIP family of channel transporters which include; the bovine major intrinsic protein (MIP), the Escherichia coli glycerol facilitator (GlpF), the peribacteroid nodulin-26 (Nod26), and the tonoplast protein from kidney bean (TIP). DiP is most similar to other plant members of this family, and in particular to the tobacco protein TobRB7 which is expressed specifically in roots. However, the expression pattern of diP suggests that its product is functionally more similar to the tonoplast intrinsic protein from kidney bean since it is most highly expressed in the cotyledons of germinating seedlings, before the cells undergo expansion growth and become photosynthetic.     

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.     

Promoter-Like Mutant with Increased Expression of the Glycerol Kinase Operon of Escherichia coli

A glycerol-specific phenotypic revertant isolated from a mutant of Escherichia coli missing enzyme I of the phosphoenolpyruvate phosphotransferase system was studied. This revertant is capable of producing higher levels of glycerol kinase and the protein mediating the facilitated diffusion of glycerol (facilitator) than wild-type cells. The kinase of the revertant is indistinguishable from the wild-type enzyme with respect to its sensitivity to feedback inhibition by fructose-1,6-diphosphate, its pH optimum, and its turnover number. The synthesis of glycerol kinase in strains bearing the suppressor locus is resistant to catabolite repression. The suppressor mutation mapped at the known glpK locus. Thus, it is suggested that the mutation occurred in the promoter of the operon specifying the kinase and the facilitator.     

Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins

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 C-terminal 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.     

Free-energy landscape of glycerol permeation through aquaglyceroporin GlpF determined from steered molecular dynamics simulations

The free-energy landscape of glycerol permeation through the aquaglyceroporin GlpF has been estimated in the literature by the nonequilibrium method of steered molecular dynamics (SMD) simulations and by the equilibrium method of adaptive biasing force (ABF) simulations. However, the ABF results qualitatively disagree with the SMD results that were based on the Jarzynski equality (JE) relating the equilibrium free-energy difference to the nonequilibrium work of the irreversible pulling experiments. In this paper, I present a new SMD study of the glycerol permeation through GlpF to explore the free-energy profile of glycerol along the permeation channel. Instead of the JE in terms of thermodynamic work, I use the fluctuation-dissipation theorem (FDT) of Brownian dynamics (BD), in terms of mechanical work, for extracting the free-energy difference from the nonequilibrium work of irreversible pulling experiments. The results of this new SMD-BD-FDT study are in agreement with the experimental data and with the ABF results.