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 Å 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. 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 Å 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. Fluid balance is fundamental to osmoregulation of a large variety of cells. Rapid transmembrane fluxes of water or glycerol are facilitated by a family of membrane channels called aquaporins or aquaglyceroporins (AQPs), 2The abbreviations used are: AQPaquaporin or aquaglyceroporinNCSnon-crystallography symmetryr.m.s.d.root mean square differencetAqpZtetrameric AqpZ. depending on their water/glycerol permeability (1Preston G.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11110-11114Crossref PubMed Scopus (725) Google Scholar, 2Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1697) Google Scholar). Structures of several AQPs have been determined, including those of human AQP1 (3Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1451) Google Scholar), Escherichia coli GlpF (4Fu D. Libson A. Miercke L.J. Weitzman C. Nollert P. Krucinski J. Stroud R.M. Science. 2000; 290: 481-486Crossref PubMed Scopus (889) Google Scholar), bovine AQP1 (5Sui H. Han B.G. Lee J.K. Walian P. Jap B.K. Nature. 2001; 414: 872-878Crossref PubMed Scopus (970) Google Scholar), and E. coli AqpZ (6Savage D.F. Egea P.F. Robles-Colmenares Y. O'Connell III, J.D. Stroud R.M. PLoS Biol. 2003; 1: E72Crossref PubMed Scopus (232) Google Scholar) as well as sheep and bovine AQP0 (7Gonen T. Sliz P. Kistler J. Cheng Y. Walz T. Nature. 2004; 429: 193-197Crossref PubMed Scopus (327) Google Scholar, 8Harries W.E. Akhavan D. Miercke L.J. Khademi S. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14045-14050Crossref PubMed Scopus (237) Google Scholar). All known AQP structures share a common molecular architecture containing four monomeric subunits assembled into a homotetrameric structure. Each monomer is characterized by an hourglass-like structure with an internal quasi-2-fold symmetry, corresponding to an intragenic gene duplication of AQP protein sequences (9Wistow G.J. Pisano M.M. Chepelinsky A.B. Trends Biochem. Sci. 1991; 16: 170-171Abstract Full Text PDF PubMed Scopus (104) Google Scholar). The functional unit of AQPs is a monomer that embraces a central water/glycerol-conducting channel surrounded by six transmembrane helices and two half-spanning helices joined end-to-end in the middle of the membrane by interlocking interactions of two highly conserved NPA tripeptide sequences. The channel lumen surface is strongly amphipathic, with oxygens and nitrogens lined up on one side and carbons on the opposite side (4Fu D. Libson A. Miercke L.J. Weitzman C. Nollert P. Krucinski J. Stroud R.M. Science. 2000; 290: 481-486Crossref PubMed Scopus (889) Google Scholar). Molecular dynamic simulations of AQPs have suggested that water molecules can coordinate each of the hydrogen-bonding groups to form a continuous single file that spans the entire length of the water-conducting channel (10de Groot B.L. Grubmuller H. Science. 2001; 294: 2353-2357Crossref PubMed Scopus (827) Google Scholar, 11Tajkhorshid E. Nollert P. Jensen M.O. Miercke L.J. O'Connell J. Stroud R.M. Schulten K. Science. 2002; 296: 525-530Crossref PubMed Scopus (766) Google Scholar). The rate of permeation is limited by two significant energetic barriers in the channel, corresponding to two channel constriction regions: one is located at the narrowest point at the channel entrance on the periplasmic side and the other at the NPA region about the quasi-2-fold axis. The narrowest constriction has been identified as the selective filter of the channel, which is formed by the side chains of Arg-189, Phe-43, and His-174 in the water-conducting channel AqpZ (6Savage D.F. Egea P.F. Robles-Colmenares Y. O'Connell III, J.D. Stroud R.M. PLoS Biol. 2003; 1: E72Crossref PubMed Scopus (232) Google Scholar) or Arg-206, Phe-200, Trp-48 in the glycerol-conducting channel GlpF (4Fu D. Libson A. Miercke L.J. Weitzman C. Nollert P. Krucinski J. Stroud R.M. Science. 2000; 290: 481-486Crossref PubMed Scopus (889) Google Scholar). These side chain differences render a narrower and more hydrophilic selective filter in AqpZ, accounting for its selectivity for water over glycerol (6Savage D.F. Egea P.F. Robles-Colmenares Y. O'Connell III, J.D. Stroud R.M. PLoS Biol. 2003; 1: E72Crossref PubMed Scopus (232) Google Scholar). Recent structural solutions of two AQP0 proteins have shown that the AQP0 water pore is much more constricted compared with those of AqpZ, AQP1, and GlpF (7Gonen T. Sliz P. Kistler J. Cheng Y. Walz T. Nature. 2004; 429: 193-197Crossref PubMed Scopus (327) Google Scholar, 8Harries W.E. Akhavan D. Miercke L.J. Khademi S. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14045-14050Crossref PubMed Scopus (237) Google Scholar). The narrowest region of the AQP0 channels is formed by an additional channel constriction in the cytoplasmic half of the pore, suggesting that additional structural components may contribute to limiting water permeation through the AQP0 channels, the rate of which is ∼40 times lower than that of AQP1 at neutral pH (12Chandy G. Zampighi G.A. Kreman M. Hall J.E. J. Membr. Biol. 1997; 159: 29-39Crossref PubMed Scopus (159) Google Scholar). aquaporin or aquaglyceroporin non-crystallography symmetry root mean square difference tetrameric AqpZ. The emerging evidence suggests that AQP channels are not constitutive conduits of water or glycerol. The crystal structures of AQP1, GlpF, and AqpZ all showed that their narrowest constrictions (selective filters) are open to water/glycerol binding, whereas the electron diffraction structure of the sheep AQP0 revealed a closed water pore. Intriguingly, the x-ray structure of bovine AQP0 exhibited a water-filled channel that contained constriction regions narrower than would be required for the passage of a water molecule. Thermal fluctuations of AQP0 side chain rotamers appeared to cause dilation of channel constrictions to larger sizes sufficient for a water molecule to pass; thus, the bovine APQ0 channel was thought to be dynamically open to water passage. This finding raised a question as to how the side chain dynamics play a role in controlling the AQP channel opening or closing. Molecular dynamic simulations of AqpZ revealed thermal fluctuations of the Arg-189 side chain between two distinct conformational states, resulting in an open or closed channel conformation (13Wang Y. Schulten K. Tajkhorshid E. Structure. 2005; 13: 1107-1118Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The thermodynamic gradient is expected to be quite shallow for this thermally activated conformational equilibrium. It is not clear whether the simulated Arg-189 thermal fluctuations could lead to steady states of channel opening and closing in milliseconds to hundreds of milliseconds, a time scale commonly observed in ion channel kinetic studies (14Katz B. Miledi R. J. Physiol. 1972; 224: 665-699Crossref PubMed Scopus (519) Google Scholar, 15Anderson C.R. Stevens C.F. J. Physiol. 1973; 235: 655-691Crossref PubMed Scopus (569) Google Scholar). In the present study, we determined the AqpZ tetrameric structure in the space group of P4122 with two tetramers/asymmetrical unit; thus, the four monomeric structures were solved independently without crystallographic averaging. Intriguingly, the tAqpZ structure revealed two distinct conformations among the four monomeric channels. One of the four channels was found in a fully open state, whereas the remaining three were closed by the side chain displacement of Arg-189 that occluded the selective filter. The tAqpZ structure therefore provides structural evidence for the presence of two distinct channel conformations during water permeation. A unique crystal packing contact between two open channels is implicated in a role of trapping the Arg-189 side chain into one of the two dynamic conformations that appear to be sufficiently stable to be resolved in the tAqpZ crystal structure. Further, Arg-189 is the only channel-lining residue in tAqpZ that shows a significant side chain displacement. This finding highlights the localization of the channel conformational dynamics to the narrowest constriction region. Expression, Purification, and Crystallization—As described previously (16Daniels B.V. Jiang J.S. Fu D. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 561-563Crossref PubMed Scopus (7) Google Scholar), His-AqpZ was overexpressed in BL21(DE3)pLysS cells and purified by one-step metal affinity chromatography. The N-terminal His tag was then removed by thrombin digestion, resulting in AqpZ that was further purified by size-exclusion high performance liquid chromatography and concentrated to ∼10 mg/ml prior to crystallization screens. The best AqpZ crystals were obtained in hanging drops by a 1:1 mix of pure AqpZ with a crystallization solution containing 16% polyethylene glycol 3350, 0.75% β-OG, 20% glycerol, 0.064 m sodium acetate, pH 4.6, 0.128 m (NH4)2SO4, 100 mm MgSO4, and 10 mm CoCl2. Data Collection and Processing—A complete x-ray dataset was collected on beam line X25 at the National Synchrotron Light Source at Brookhaven National Laboratory. The dataset was indexed, integrated, and merged using the software package HKL2000 (17Otwinowski Z. Minor W. Methods Enzymol., Macromol. Crystallogr. 1997; 276: 307-326Crossref Scopus (38617) Google Scholar). Because of significant x-ray beam variations during data the dataset was into three and and then merged to an of to 3.2 Å with an of for the and structure was solved by molecular using a tAqpZ by symmetry of a GlpF Acta Crystallogr. Sect. A. PubMed Scopus Google Scholar) revealed a symmetry to an of two unit by a The was using the software package G.M. P. Jiang J.S. J. M. T. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar). the first of the was and used as a for further with one tAqpZ as a structures of two are several of and water molecules were identified and into the The and difference density were used the Acta Crystallogr. Sect. A. Scopus Google Scholar). All were using the software package of the tAqpZ data to 3.2 Å were collected AqpZ crystals to space group P4122 with two by a to the crystallographic The structure was solved by molecular and to an of and of without between two to of root mean square differences to by of the a was to the structural an of and of this of several strong electron were on the surface of the The and of electron were of and The of and were further to and by water molecules were to a of and of The tAqpZ with two molecules and six accounting for electron found on the surface as well as in the of the AqpZ The water electron were observed four monomeric channels, and D and where is the of the for where and are observed and structure is a of and is over the remaining of in are in the in protein all in a The of two in the unit showed of the the crystallographic additional not structural suggesting that are structural differences among the four AqpZ of a of Å among four monomers, with differences in the where crystal were between AqpZ differences were obtained the AqpZ monomer structures solved previously in a space group of with two structural differences were between two in the AqpZ crystals (6Savage D.F. Egea P.F. Robles-Colmenares Y. O'Connell III, J.D. Stroud R.M. PLoS Biol. 2003; 1: E72Crossref PubMed Scopus (232) Google Scholar). of the monomeric structure of AqpZ six transmembrane helices and two half helices in a that a symmetry in the membrane as described previously (6Savage D.F. Egea P.F. Robles-Colmenares Y. O'Connell III, J.D. Stroud R.M. PLoS Biol. 2003; 1: E72Crossref PubMed Scopus (232) Google Scholar). The AqpZ channel of a periplasmic and a cytoplasmic vestibule, in and by a central pore Å in The hydrophilic provides two of carbonyl each by four chain of Thr-183, and the periplasmic side and those of quasi-2-fold and the cytoplasmic chain is the highly and NPA about the quasi-2-fold where and groups of their side chains to the channel. This complete of carbonyl oxygens and are on the hydrophilic to form a chain of and for of water thus a water-conducting the of the channel, an of and provides to the of the pore. The channel two highly conserved the selective filter and the NPA region. ∼10 Å the NPA on the periplasmic side is the selectivity filter formed by the side chains of Arg-189, and the carbonyl of on the hydrophilic and the of on the opposite in AqpZ of electron were observed in each of the AqpZ monomer channels The electron were located in the periplasmic half of the central pore between the selective filter and the NPA resulting in of several water the of water molecules and their are among the four monomer channels. In the selective filter are two water molecules in monomer one in monomer and water observed in and D of and not with is the selective where is to the of the Arg-189 guanidino group of the selective a tridentate H-bond with the of the His-174 group of Arg-189 guanidino group and chain carbonyl oxygen is the selective filter by the carbonyl oxygen and the Arg-189 In the NPA are two water molecules and in monomer three in monomer two in monomer and one in monomer D These water molecules the NPA to the cytoplasmic vestibule, the are present in and a H-bond with of or The middle are present in all four and to carbonyl oxygen The water is only in monomer with an H-bond with the carbonyl oxygen of The of water molecules in all four are with a with their oxygen the of the channel as described previously E. Nollert P. Jensen M.O. Miercke L.J. O'Connell J. Stroud R.M. Schulten K. Science. 2002; 296: 525-530Crossref PubMed Scopus (766) Google Scholar). Arg-189 in the and groups in the four monomer channels are in the with one marked exception in the selective filter where the side chain of Arg-189 was found in two distinct conformational orientations. on Arg-189 of a difference in the of the guanidino group of Arg-189 in the monomer A as compared with those in and which are identical In monomer the Arg-189 guanidino group is orientated to the length of the channel, an opening in the selective filter for of a water This Arg-189 is identical to those of Arg-189 observed in all of the AQP x-ray structures determined thus In and the Arg-189 guanidino group bends over toward the carbonyl oxygen of to form an and up the selective filter two of the Arg-189 side we Arg-189 side chains between A and removed water molecules the and and simulated Arg-189 side chain in A and to their we the Arg-189 side chains in monomer A with several rotamers and found that rotamers on a single by and in the of water Arg-189 rotamers in and on the of the for in Arg-189 were in the of for monomer for monomer for monomer and for monomer D. The of Arg-189 in and D were to the tAqpZ whereas binding of water molecules to A and the Arg-189 to the Therefore, the two of the Arg-189 guanidino The observed side chain displacement was unique to Arg-189, side chain of all other residues were nearly Further, we only observed two distinct Arg-189 side chain conformations among four monomer channels. This was in with molecular dynamic simulations of AqpZ that Arg-189 side chain could only one of two distinct as to conformations (13Wang Y. Schulten K. Tajkhorshid E. Structure. 2005; 13: 1107-1118Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). in the how the Arg-189 in monomer A could be stabilized to a conformation those in and we the difference in crystal packing between monomer A and D. shown in are three of in the and at the of two AqpZ an unit and a contact between two The is formed by an in the of two monomer A thus, monomer A is more in the only to the whereas monomer and D are the The surface at the and are and A of the contact revealed a strong electron density that two of in the first transmembrane helices The of the electron density was of a we were not to a molecular at the The contact point at is to the highly conserved which two with chain to four chain of and on the channel lumen The carbonyl of highly conserved with of as a of the interlocking that the two NPA sequences The of highly conserved with the chain of Arg-189 is to through a chain of highly conserved interactions The of this chain of identical among the four suggesting the of to the interlocking It that the side chain of Arg-189 is the at the of this chain of which may the of crystal packing contact at to be to this unique crystal packing contact between two A is correlated with the open conformation of this in the are among four is to how differences in may contribute to of the Arg-189 All the known AQP crystal structures thus have been determined with crystallographic of monomeric structures to crystal packing The tAqpZ structure a unique tetrameric solution that the of two distinct Arg-189 side chain in the filter. This is with molecular dynamic that suggested the presence of two distinct the Arg-189 side chain in molecular dynamic simulations appeared to be in rapid between two distinct conformations only by thermal In the of the Arg-189 side chains in the tAqpZ structure were or lower than the protein of Further, the on Arg-189 exhibited distinct side chain of two of the Arg-189 guanidino group at a limited of Arg-189 conformations to one of the two distinct of the These that the of two distinct Arg-189 conformations in tAqpZ crystals is It is not whether the two Arg-189 conformations to open and closed states of an AqpZ channel or whether two dynamic conformations found during water permeation through the narrowest constriction of the channel. kinetic of opening and closing of AqpZ channels by single channel the crystal structure of tAqpZ only provides as to the of the Arg-189 The structural difference between the and conformation is to the Arg-189 side chain orientations. This of a more protein conformational suggests a the two Arg-189 conformational the observed two Arg-189 conformations are by thermal The thermodynamic between two Arg-189 conformations is to the open probability of the water pore, whereas the shallow thermodynamic gradient between the two Arg-189 conformations may be to the AqpZ channel into one of two states without additional The Arg-189 in tAqpZ crystals appeared to be into two distinct stable conformational states by asymmetrical crystal packing AQP crystal structures where was or packing with the transmembrane the tAqpZ structure reveals interactions in the transmembrane and that asymmetrical crystal packing are correlated with differences in Arg-189 side chain The conformations of four Arg-189 side chains are stabilized whereas of other side chains are identical in four monomer channels. This finding highlights the dynamic of the Arg-189 side chain in a stable protein The unique Arg-189 conformation in monomer A is to a crystal packing at in the first transmembrane Because the molecular dynamics that the of Arg-189 are in between two thermally activated states, is that a may be to Arg-189 in one of the two It is that interactions at may the of Arg-189 conformations through a chain of highly differences other asymmetrical crystal packing may contribute to of the Arg-189 in to ion channels among open and closed conformational The opening or closing of an ion channel in flow of in an an gradient across the channel. channel opening and closing can be by single channel Because water/glycerol permeation through AQP channels is not to high crystal structures may be used to the opening or closing of the water-conducting pore. A of an open channel of one or several binding in a transmembrane that are of the membrane at the time P. J. Membr. Biol. PubMed Scopus Google Scholar). The Arg-189 conformation observed in monomer A a water molecule binding to the narrowest constriction of the thus, monomer A is in an open channel The of the closed channel conformation is not The water pore structure in the bovine AQP0 was to be for water side chain movements could sufficiently the channel constrictions for water passage W.E. Akhavan D. Miercke L.J. Khademi S. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14045-14050Crossref PubMed Scopus (237) Google Scholar). into membrane channels may be by a of by barriers P. 16: PubMed Scopus Google Scholar). permeation through membrane channels may a of thermally activated between of Because the barriers in AQP0 could be by thermal fluctuations of side chain AQP0 could be a dynamically open channel. the other asymmetrical crystal in tAqpZ and D into a stable conformation in which of Arg-189 bends over to form a strong with the chain carbonyl oxygen of in the thus the narrowest constriction of the channel not for water The chain between and Arg-189 was shown to be stable of the Arg-189 Molecular dynamic simulations of the of the AqpZ channel that the of the selective filter by Arg-189 could lead to a complete of the water pore (13Wang Y. Schulten K. Tajkhorshid E. Structure. 2005; 13: 1107-1118Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). These that the Arg-189 conformation in and D may an for thermally activated water permeation. and D are in a closed channel stabilized in by asymmetrical subunit interactions of crystal packing in AqpZ may with regulating the water by the of open or closed channels in an AqpZ The for a conformational to be in shown in the closed is by the through the carbonyl oxygen and chain the Arg-189 guanidino group an H-bond with the chain carbonyl which is to the of the chain the of the through a side chain to the chain In the channel open state, the Arg-189 oxygen H-bond is by two with two water to Arg-189 and to to Arg-189 thus the through a of by the carbonyl and chain Therefore, the Arg-189 conformational between the open and closed is associated with two between Arg-189 and In the are stabilized by an This finding the of the to the Arg-189 side chain dynamics and provides a as to the of only two distinct Arg-189 as to Intriguingly, as the quasi-2-fold contribute to Arg-189 conformational dynamics through a chain of by the interlocking Arg-189, and are among the conserved residues in the AQP protein The high of to their functional M. Synchrotron Light Brookhaven National for in x-ray diffraction