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.     

Water transport in aquaporins: osmotic permeability matrix analysis of molecular dynamics simulations

Single-channel osmotic water permeability (p(f)) is a key quantity for investigating the transport capability of the water channel protein, aquaporin. However, the direct connection between the single scalar quantity p(f) and the channel structure remains unclear. In this study, based on molecular dynamics simulations, we propose a p(f)-matrix method, in which p(f) is decomposed into contributions from each local region of the channel. Diagonal elements of the p(f) matrix are equivalent to the local permeability at each region of the channel, and off-diagonal elements represent correlated motions of water molecules in different regions. Averaging both diagonal and off-diagonal elements of the p(f) matrix recovers p(f) for the entire channel; this implies that correlated motions between distantly-separated water molecules, as well as adjacent water molecules, influence the osmotic permeability. The p(f) matrices from molecular dynamics simulations of five aquaporins (AQP0, AQP1, AQP4, AqpZ, and GlpF) indicated that the reduction in the water correlation across the Asn-Pro-Ala region, and the small local permeability around the ar/R region, characterize the transport efficiency of water. These structural determinants in water permeation were confirmed in molecular dynamics simulations of three mutants of AqpZ, which mimic AQP1.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Selectivity and conductance among the glycerol and water conducting aquaporin family of channels

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 file throughout the 28 A long channel in AqpZ. The basis for absolute exclusion of proton or hydronium ion conductance through the line of water is explored using simulations.     

Visualization of AqpZ-Mediated Water Permeability in Escherichia coli by Cryoelectron Microscopy

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene, aqpZ, in Escherichia coli. By employing cryoelectron microscopy to compare E. coli cells containing (AqpZ+) and lacking (AqpZ-) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.     

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity

Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.     

Water transport and ion-water interaction in the gramicidin channel

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.     

Population Shift between the Open and Closed States Changes the Water Permeability of an Aquaporin Z Mutant

Aquaporins are tetrameric transmembrane channels permeable to water and other small solutes. Wild-type (WT) and mutant Aquaporin Z (AqpZ) have been widely studied and multiple factors have been found to affect their water permeability. In this study, molecular dynamics simulations have been performed for the tetrameric AqpZ F43W/H174G/T183F mutant. It displayed ～10% average water permeability compared to WT AqpZ, which had been attributed to the increased channel lumen hydrophobicity. Our simulations, however, show a ring stacking between W43 and F183 acting as a secondary steric gate in the triple mutant with R189 as the primary steric gate in both mutant and WT AqpZ. The double gates (R189 and W43-F183) result in a high population of the closed conformation in the mutant. Occasionally an open state, with diffusive water permeability very close to that of WT AqpZ, was observed. Taken together, our results show that the double-gate mechanism is sufficient to explain the reduced water permeability in the mutant without invoking effects arising from increased hydrophobicity of the channel lumen. Our findings provide insights into how aquaporin-mediated water transport can be modulated and may further point to how aquaporin function can be optimized for biomimetic membrane applications.     

Solid-state NMR chemical shift assignments of aquaporin Z in lipid bilayers

Aquaporin Z is the first identified prokaryotic water channel in Escherichia coli with a high water permeability and strict substrate selectivity. Here we report nearly complete (94% of amino acid residues) ¹³C and ¹⁵N chemical shift assignments of AqpZ reconstituted in the lipid bilayers using a set of 2D and 3D magic angle spinning solid-state NMR spectra. Secondary structure of AqpZ predicted from chemical shift assignments is generally similar to that of X-ray structure with a number of differences in loop and near-loop regions. The BMRB accession number of the assignments is 27244.     

Electrostatic tuning of permeation and selectivity in aquaporin water channels

Water permeation and electrostatic interactions between water and channel are investigated in the Escherichia coli glycerol uptake facilitator GlpF, a member of the aquaporin water channel family, by molecular dynamics simulations. A tetrameric model of the channel embedded in a 16:0/18:1c9-palmitoyloleylphosphatidylethanolamine membrane was used for the simulations. During the simulations, water molecules pass through the channel in single file. The movement of the single file water molecules through the channel is concerted, and we show that it can be described by a continuous-time random-walk model. The integrity of the single file remains intact during the permeation, indicating that a disrupted water chain is unlikely to be the mechanism of proton exclusion in aquaporins. Specific hydrogen bonds between permeating water and protein at the channel center (at two conserved Asp-Pro-Ala "NPA" motifs), together with the protein electrostatic fields enforce a bipolar water configuration inside the channel with dipole inversion at the NPA motifs. At the NPA motifs water-protein electrostatic interactions facilitate this inversion. Furthermore, water-water electrostatic interactions are in all regions inside the channel stronger than water-protein interactions, except near a conserved, positively charged Arg residue. We find that variations of the protein electrostatic field through the channel, owing to preserved structural features, completely explain the bipolar orientation of water. This orientation persists despite water translocation in single file and blocks proton transport. Furthermore, we find that for permeation of a cation, ion-protein electrostatic interactions are more unfavorable at the conserved NPA motifs than at the conserved Arg, suggesting that the major barrier against proton transport in aquaporins is faced at the NPA motifs.     

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.     

Hydroxide and proton migration in aquaporins

Hypothetical hydroxide and proton migration along the linear water chain in Aquaporin GlpF from Escherichia coli are studied by ab initio Car-Parrinello molecular dynamics simulations. It is found that the protein stabilizes a bipolar single file of water. The single file features a contiguous set of water-water hydrogen bonds in which polarization of the water molecules vary with position along the channel axis. Deprotonation of the water chain promotes the reorientation of water molecules while the hydroxide ion rapidly migrates by sequentially accepting protons from the neighboring water molecules. The hydroxide ion is not attracted by a conserved, channel-lining arginine residue, but is immobilized at two centrally located, conserved Asparagine-Proline-Alanine motifs where fourfold coordination stabilizes the ion. Hydroxide transition from the channel vestibules into the channel lumen is strongly influenced by electrostatic coupling to two conserved oppositely aligned macrodipoles. This suggests that the macrodipole's negative poles play a role in preventing hydroxide ions from entering into the channel's inner vestibules. Water protonation within the lumen facilitates water reorientation and subsequent proton expelling occurs. In the periplasmic half-channel, expelling occurs via the Grotthuss mechanism. Protonation within the cytoplasmic half-channel implies wire-breakage at the Asn-Pro-Ala motifs. The proton is here diffusively rejected as (H(5)O(2))(+).     

Water permeation dynamics of AqpZ: a tale of two states

Molecular dynamics simulations of aquaporin Z homotetramer which is a membrane protein facilitating rapid water movement through the plasma membrane of Escherichia coli were performed. Initial configurations were taken from the open and closed states of crystal structures separately. The resulting water osmotic permeability (pf) and diffusive permeability (pd) displayed distinct features. Consistent with previous studies, the side chain conformation of arginine189 was found to mediate the water permeability. A potential of mean force (PMF) as a function of the distance between NH1 of R189 and carbonyl oxygen of A117 was constructed based on the umbrella sampling technique. There are multiple local minima and transition states on the PMF. The assignment of the open or closed state was supported by the permeability pf, calculated within trajectories in umbrella sampling simulations. Our study disclosed a detailed mechanism of the gated water transport.     

Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions

Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped-flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.Results. The kinetics of plasmolysis or deplasmolysis of Escherichia coli cells in suspension, in response to osmotic challenges, was analysed by stopped-flow spectrophotometry. Cytoplasmic volume variations were monitored either by GFP (green fluorescent protein) fluorescence quenching or by 90 degrees scattered light. The single exponential response to up-shocks in the impermeant solute mannitol was strongly accelerated when the cells expressed the native E. coli AQP AqpZ (rate constant 37.24 versus 3.05 s(-1) for control cells). The responses to hyperosmotic shocks realized with glycerol were biphasic. First, a light-scattering increase corresponded to cell plasmolysis. Secondly, deplasmolysis occurred when glycerol entered into the cell. Both phases were accelerated when the aquaglyceroporin GlpF was present in cell membranes. We concluded that the behaviour of MIP-expressing bacteria in the stopped-flow system was qualitatively identical with that reported for MIP-expressing oocytes or MIP-containing proteoliposomes. We then used this system to analyse the effects of mutations in the pore constriction of Gla(Llac), the aquaglyceroporin from Lactococcus lactis. In the present study, we show that Gla(Llac) loses its ability to transport glycerol but retains its ability to transport water when Val(223) was replaced by a histidine, the residue at the equivalent position in strict AQPs.Conclusions. These results show that stopped-flow spectrophotometry performed on E. coli cell suspensions is a useful experimental system to analyse the selectivity of wild-type or mutant MIP proteins and that a bifunctional aquaglyceroporin switches to an AQP by a single amino acid mutation in the pore constriction.     

Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel

AqpZ is a homotetramer of four water-conducting channels that facilitate rapid water movements across the plasma membrane of Escherichia coli. Here we report a 3.2 angstroms crystal structure of the tetrameric AqpZ (tAqpZ). All channel-lining residues in the four monomeric channels are found orientated in nearly identical positions with one marked exception at the narrowest channel constriction, where the side chain of a highly conserved Arg-189 adopts two distinct conformational orientations. In one of the four monomers, the guanidino group of Arg-189 points toward the periplasmic vestibule, opening up the constriction to accommodate the binding of a water molecule through a tridentate H-bond. In the other three monomers, the Arg-189 guanidino group bends over to form an H-bond with carbonyl oxygen of the Thr-183, thus occluding the channel. Therefore, the tAqpZ structure reveals two distinct Arg-189 confirmations associated with water permeation through the channel constrictions. Alternation between the two Arg-189 conformations disrupts continuous flow of water, thus regulating the open probability of the water pore. Further, the difference in Arg-189 displacements is correlated with a strong electron density found between the first transmembrane helices of two open channels, suggesting that the observed Arg-189 conformations are stabilized by asymmetrical subunit interactions in tAqpZ.     

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.     

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.

     

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.     

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.     

Microscopic model for selective permeation in ion channels.

Ionic permeation in the selectivity filter of ion channels is analyzed by a microscopic model based on molecular kinetic theory. The energy and flux equations are derived by assuming that: (a) the selectivity filter is formed by a symmetrical array of carbonyl groups; (b) ion movement is near the axis of the channel; (c) a fraction of water molecules is separated from the ion while it moves across the selectivity filter; (d) the applied voltage drops linearly across the selectivity filter; (e) ions move independently. Energy profiles, single channel conductances, and the degree of hydration of K+ in a hypothetical K+ channel are examined by varying the following microscopic parameters: ion radius and mass, channel radius, number of effective water dipoles, and number of carbonyl groups. The i-V curve is linear up to +/- 170 mV. If the positions of energy maxima and minima are fixed, this linear range is reduced to +/- 50 mV. Channel radius and ion-water interactions are found to be two major channel structural determinants for selectivity sequences. Both radius and mass of an ion are important in selectivity mediated by these interactions. The theory predicts a total of 15 possible kinetic selectivity sequences for alkali cations in ion channels with a single selectivity filter.