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articles Structural basis of waterspecific transport through the AQP1 water channel llaixin Sui BongGyoon llani John K Lew Peter Walian amp Bing K Jari Life Sciences Division Lawrence Berkeley National Laboratory and 1 Graduate Group in Comparative Biochemistry University of California Berkeley California 94720 USA T These authors contributed equally to this Work Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients These channels are believed to be involved in many physiological processes that include renal water conservation neuro homeostasis digestion regulation of body temperature and reproduction Members of the water channel superfamily have been found in a range of cell types from bacteria to human In mammals there are currently 10 families of water channels referred to as aquaporins AOP AOPOAOPQ Here we report the structure of the aquaporin 1 AOP1 water channel to 22A resolution The channel consists of three topological elements an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter Within the selectivity filter four bound waters are localized along three hydrophilic nodes which punctuate an otherwise extremely hydrophobic pore segment This unusual combination of a long hydrophobic pure and a minimal number of solute binding sites facilitates rapid water transport Residues of the constriction region in particular histidine 182 which is conserved among all known waterspecific channels are critical in establishing water specificity Our analysis of the AOP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable AQPO AQP9 can be divided into two major groups AQPO AQPZ AQP4 AQP6 and AQP8 permeable to water but not to small organic and inorganic molecules3 and AQP3 AQP7 and AQP9 permeable to glycerol or urea as well as water Both groups of channels are active in many physiological functions AQPI relative molecular mass M r 28000 was initially found in red blood cells and renal proximal tubules AQPI water channels allow water but not ions including protons to move freely and bidirectionally across the cell membrane Sequence analysis shows high homology among members of the AQPI family and that the two halves of the sequence exhibit a high degree of similarity Each sequence half contains an NPA asparagine pro line alanine motif which is conserved throughout the AQP superfamily including the glycerol facilitators Moderate resolution projection and low resolution 3D three dimensional maps of AQPI derived from electron crystallographic studies provided the rst structure based evidence for a general architecture consisting of six helices surrounding two putative helical structures within the membrane bilayerf 1 Models of AQPI derived from electron crystallographic structural studies at about 4A resolution have recently been reported they independently con rm the presence of two membrane inserted non membrane spanning helices12 14 We eport here the structure of AQPI from bovine red blood cells at 22 A resolution as determined be ray crystallography Table 1 At this resolution the positions of the side chains that establish the properties of the transmembrane channel pathway as well as water molecules captured in transit are clear Critical differences exist between this AQPI structure and those determined by electron crystallography particularly in the pore pro les12 14 For example the constriction region in models derived from these previous studies is approximately 8A away from this region in the model of AQPI presented here The constriction region in our model is also much smaller than that inferred from the high resolution structure of the Escherichia coli glycerol facilitator GlpF The high resolution structure of AQPI now reveals the structural basis for water speci city together with the structure of GlpF it also 872 2001 Macmillan Magazines Ltd provides the molecular details necessary to understand the mechan isms regulating water and other solute selectivity within the aqua porin superfamily Architectural overview The functional unit of AQP1 is a tetramer with each monomer providing an independent water pore Fig 1a c Each monomer contains six transmembrane helices packed to form part of a trapezoid like structure when viewed normal to the membrane Table 1 Data collection and refinement statistics Data set Resoldtlon A Complete Ram J u Au AQPl 2 2 2 so 2 20 99 7 too 0 5 258 2 41 a 5 a Tl AQPl M 0 97871 2 a 2 85 2 80 99 2 99 a 5 5 Al 8 50 o 9 7 x2 0 97954 2 a 2 85 2 80 99 2 too 0 5 o 29 2 5a 513 8 as t 00800 2 a 2 85 2 80 99 4 too 0 5 1 so 2 54 513 5 M 0 95593 2 a 2 85 2 80 99 2 99 a a a 47 9 45 a a a Re nement statlstlcs Resoldtlonoi l5 2 2 Reflectlons ln worklngtest set 169341235 R J 26 6 Rtree J 30 8 NonglyClne reslddes ln most favourable reglon of 97 2 Ramacnandran plot Odtllers and generously allowed reglons 2 8 r m s deVlatlortfrom ldeallty Bond lengths A Bond angles degrees 0 0066 l 25 AurAQPl lndlcates tne data set collectedlrom crystals grown lntne presence ol gold Cyanlde and TlePl denotes tne data ootalned lrom lhalllumrderlvallzed Aopl crystals The labels mew lndlcate tne dlrlerent data sets collected lor multlrwavelenglh anomalous dlllractlon MAD pnaslng The wavelengtns used are snown ln parentneses Thls large Rm value ls tne consequence olslgnlllcant anlsotropv wnen tne data were llltered at a lo level tne percentage ol rellectlons removed lrom tne quadrants ol maxlmum anlsotropv was proportlonallv tne nlgnest and tne lollowlng values were ootalned R9 349 completeness was 78 0 andlo 57 R9 Ell bl21 wnere llstne measurement ollntensltv R lemS Paul2F lor all values and PM ls tne R value lor tne rellectlons tnat were excluded ln rellnemenl wnere Fm ls tne ooserved natlve amplltude and PW ls tne one calculated lrom atomlc models The values lor tne nlgnest resolutlon zone are snown ln parentneses NATURE l VOL 414 l 2027 DECEMBER 2001 wwwmturecom plane Two membrane inserted but non membrane spanning helices which de ne a major portion of the pore are partially enclosed bythis structure and form an integral part of the outer wall as well This general folding topology is similar to that reported for the structure of E coli GlpF S and to those reported from electron crystallographic studies ofAQPl refs 12 14gt Residues comprising the six transmembrane and the two membrane inserted non membrane spanning helices M1 M8 are depicted in Fig 1b The amino terminus of the molecule is located at the cytoplasmic side ofthe cell membrane as determined from earlier biochemical studies and leads into the rst oftwo transmembrane helices Thesetwo helices M1 M2 are followed by a membrane inserted loop that contains an NPA motif and leads mm mm W apr WM MGM m min apr mm mm w W apr Figure l Modelsot Allin seduence alignment of selected supertamily members and a View of tne density map a Combined ribbon diagram and space tilling model of Allin monomeruiewed parallel totne membrane nAmino acid seduencealignmentot bouine Allin numanAuin E mirAdpz humanAQPBandEcl7 Glprroduceduslllg CLUSTAL we Allin membrane embedded nelicesare colour coded as snown in a and labelled Ml M8 M3 and M7 are membrane inserednon membrane spanning nelices Residues lining tne extended narrowpore selectlvllyfllterof bouineAuin are indicated in red italicized letters o Allin tetrameruiewed normal to tne membrane two ottne maneuver m l2n27 oecmeee zooi lwwwnaruresom to 2001 Macmillan Magazines Lid into the non membrane spanning helix M3 The lo op exiting this very short helix enters the cytoplasm and turns back into the membrane to connect to transmembrane helix M4 which ends the N terminal half ofthe structure on the extracellular side ofthe cell membrane The second half of the structure is essentially an inverted repeat ofthe topology ofthe rst half and results in the location of the carboxy terminus at the cytoplasmic face Three nonylglucoside detergent molecules have been located on a region ofthe monomer surface in contact with the extracellular lea et of the lipid bihyer Each monomer is 4oii across and 5oii long Structure of the pure The pore ofAQPl is dumbbell like in shape when viewed in pro le nni itawaerianimrrpr u gr nxivxmm raneiggpiirrny ojysmy am M3 51 use rwu M Xe etoi tsge gifn i iilit it isl i lg E v f ib r hg mii lm nevmxxnseev seeaitraygrainiragrnmvrs i3 n 39 v AXV 391 de Ln 1 L v L 39 2 iii y aiy aimi its r in 39 Gr m quotr ia V39L gi i i 39i lnligng i fi an or in quot ryspya amiaauvi itiuiee r mry Es Wastes glean 1 M5 M6 mime min v era margin 1 xmxgqng nvnavnngm aeiafSioyaigiiam m uiaauyyeivu myiu M amipo aeraibiaminiisieyg w mayguyrlrariupiriaign j i il liilliliF egei 3 M7 ins M u i i i li iiw iii tili i 1 NEW A a inseparaan aye bnauseaenrorryeanisonii iyam nnvg win an new 2r monomers alsocontain a space tilling modelto snowtne pore entrance ine black arrow points along tne direction of tne cut line usedto produce tne monomeruiew in Fig 2b a Stereouiewottne 2io 7 Fc electron density map l Eocontoul and tne corresponding region of tne Allin model ine View is from the extracellular side down tne pore and centred abouttne constriction region ine spnerical densities near tne pore centre are tnose of tne two water molecules located in tne extracellular naltot tne selectiuity tilter ine tigure was produced using lllltscnlin36 and Rastede s13 articles Fig 2a b consisting ofthree general elements an extracellular vestibule an extended narrow pore or selectivity lter containing the constriction region and a cytoplasmic vestibule Helices M4 and M8 do not contribute residues to the pore Abouthalfofthe channel wall along the selectivity lter can be considered hydrophobic and the other half hydrophilic The hydrophilic face provides the chemical groups that are essential for displacing certain waters of hydration and therefore establishes a pathway for coordinating water transport The steep crossing angles of the monomer helices aid in the formation of the extracellular and cytoplasmic vestibules Shaping of the vestibule mouths is completed by loop regions at each monomer face and in addition by the N and C terminal residues at the cytoplasmic face Fig 1a c The extracellular vestibule is roughly conical in shape with a mouth diameter ofabout 15 A pore size estimates are based on the van der Waals radii used in the AMBER program suite The population of polar moieties along the surface ofthe extracellular vestibule consists predominantly of polar residues only a small number ofwhich are charged and polar groups from the solvent exposed backbone of extended loop regions Over a distance of about 20A the extracellular vestibule tapers down to its narrowest point 8A in diameter forming the constriction region and the beginning ofthe 20 A long selectivity lter Fig 3 A series ofsolvent accessible carbonyl oxygens forms a path leading from the extracellular vestibule through the constric tion regions ofboth AQPl and GlpF in the case ofbovine AQPl these groups are provided by residues G190 c191 G192 and 1193 of Extracellular Cytoplasmic Figure 2 slue ylews ofAQPl a Backbone ln nbbon format resluues ueplceu ln ball anu 5le representatlon resluues lllllllg tne pore are snown ln opauue colours lne pore proflle ls nlpnllpnteu by an array of blue oots generated by me program HOLE lne constrlctlon reglon lsylslble as me plncneu ln area ln tne extracellularnalfofllle proflle n slue ylewcutaway ofa space fllllng mouel maue along tne axls lnulcateu by me blackalmw ln Flo lc Resluues not ln ulrectcontaclwltn tne pore are dark grey lne pore s14 9 2001 Macmillan Magazines Ltd the connecting loop leading into non transmembrane helix M7 Fig 4 This architectural scheme which positions a helix linker loop so as to form a key element of the selectivity lter is reminiscent of the structural motif employed in the potassium channel from Streptomyres lividmls KcsA for the removal ofsolute hydration waters At the constriction region residues H182 and R197 along with the solvent accessible carbonyl oxygen of residue c191 form the hydrophilic face ofthe pore Fig 5 The imidazole ring ofH182 is fully extended into the pore while the bulk of R197 is pointed upwards almost parallel to the pore axis in a manner similar to that observed for the equivalent arginine ofthe GlpF channel Opposite the hydrophilic face at the constriction region is the hydrophobic face de ned here by residue F58 Three ofthe four residues de ningthe constriction region ofthe AQPl pore R197 H182 and F58 are conserved across the water speci c aquaporinsm Access to the carbonyl group of the fourth residue C191 seems to be essential at that location The conserva tion of arginine histidine and phenylalanine side chains at their respective locations within the constriction region in the known water channels is a strong indicator of channel water speci city and may be useful in assigning function to the sequences of candidate aquaporins forwhich physiological assays have notbeen performed In GlpF the H182 ofAQPl is replaced by glycine This substitu tionprovides the additional room needed to accommodate a second substitution phenylalanine for C191 Fig 5 These substitutions have two critical effects on the characteristics of the GlpF oonstric tion region they increase its size and its hydrophobicity lllllllg ln lllelopflgllle conslstspnmanlyofpolargroupsulstrlbuteualonptne lenptn oflne selectlylly nlter tne complementary llalf ls preuomlnantly nyuropnoblc ln nature lne constrlctlon reglon anu pseuuotwo foldaxls centreu abouttne NPA mollfs are lnulcateu by blue anu blackanows respectlyely1ne pore proflle blue uots ls supenmposeu on eacn cnannel nalf1neflgure was produced Uslng lnlltsmllll39 anu RasteralJ NAmulvm AM lzn27 DECEMBERZBBI wwwnamecom Past the constriction region the pore opens up again averaging about 4A in diameter over the next 15A About 8A past the constriction point residues from the two highly conserved NPA motifs are brought into close proximity owing to the end to end packing of the two short membrane inserted non membrane spanning helices M3 and M7 This places the terminal amine groups of the two NPA motif asparagine residues N194 and N78 in the pore The pseudo two fold axis ofthe molecule runs parallel to the membrane plane and is located approximately halfway between these two short helices As in the case of Glp F short helices M3 and M7 are situated end to end and lengthwise along one side of the channel so that the positive ends oftheir helical dipoles point inward toward the pore The aquaporins therefore differ from KcsA in their use ofsuch non membrane spanning helices in KcsA four such helices are posi tioned radially around the pore axis and the direction ofthe dipoles in reversed establishing a net negative zone in the middle of the pore The two helix aquaporin motif is found in both water and glycerol selective channels which suggests that this structure is not used in discriminating between water and glycerol The examples of KcsA and the aquaporins suggest thatpore located non membrane spanning helices may serve as functionally critical structural motifs in other classes of transport associated membrane proteins A second string of carbonyl oxygens lines the pore this time extending away from the NPA motifs towards the cytoplasmic vestibule and are provided by residues L77 H76 A75 and G74 ofthe connecting loop from M2 into the other membrane inserted non membrane spanning helix M3 Fig 4 The complete set of pore accessible carbonyl oxygens and asparagine amine groups are AQPl Ettecliye pore diameter A l Hydrophobl 1L c n rls L Relative position A Figure alne ettectiye pore diarneter a and nydropnobicity n ot tne AllPl and GlpF cnannels Green and dark blue arrows indicate tne locations ottne constriction region and tne pseudo two told axis respectively Below tnese arrows a lignt blue bar indicates tne location ot tne extracellularyestibule a red bar tne selectiyity tilterand a green bar tne cytoplasrnic vestibule lne tnree black bars in tne nydropatny protile identity nydropnilic nodeswitnin tne selectiyitytilter Pore diarn eters were detenninedwitn AMBER basedyan derWaals radii Band analysed using tne prograrn HOLE Hydropnobicity was cnaracterized using tne Kyle and Doolittle arnlno acid nydropatny scale39andatour point averaging window across tne pore lining residues NATUEElVOE 414 l2n27 DECEMBER zuui lwwwnarmeiom 9 2001 Macmillan Magazines Ltd arranged in a long pitched helical pattern forming much of the hydrophilic half ofthe selectivity filter Figs 2b and 4 A similar distribution of pore lining groups was reported for the structure of GlpF ref 15 Near the cytoplasmic end of the selectivity filter is another pore accessible histidine H76 Unlike H182 this histidine is highly conserved across the aquaporin superfamily and glycerol facilita tors Instead of extending maximally into the pore as does the constriction defining H182 H76 is fixed against the side ofthepore wall The GlpF counterpart to H75 is oriented within the pore in a similar fashion In the last 8 10A of the channel the pore flares out to form a cytoplasmic vestibule with a mouth approximately 15 Awide As its walls are somewhat more uniform in height this vestibule is more conical in shape than the extracellular vestibule and its mouth is essentiallyperpendicular to the pore axis Here the concentration of polar residues increases significantly from that occurring in the selectivity lter these residues establish a zone within the inner half of this vestibule that is more hydrophilic than that of GlpF Fig 3 Location of waters in the channel Waters have been identi ed at four locations within the AQPl selectivity filter Fig 4 The density attributable to a single water molecule is located adjacent to the constriction region about 7A from the pseudo two fold axis towards the extracellular surface At this location water is coordinated by hydrogen bonds established with the e2 nitrogen ofH182 and the backbone carbonyl oxygen of G192 The next two waters are centred about the pseudo two fold Extracellular 3190 r i H t R197 39 quot x V CWQLJF TleBZ r t E fttme 91P Nmzpquot has i 4 l l gLNyg 7 c sr r i K77 s i 9 i r A75 5VGH75 L M 5 r 374 l 39 v i A Cyroplasrnic v u FiguredSeleCtlilty tilterwaterrnoleculesand residues tonning tne nydropnilic tace ottne cnannel pore Cutaway side yiew ottlie cnannel witn secondary structure snown in ribbon torrnat side cnainscritical to establisning tne long pitcned nelical nydropnilc patli across tne lengtn ottlie selectiyity tilterare snown ine tourwater rnolecules located witnin tne selectiyitytilterare depictedas green spneres 0t tnese tourwater rnolecules only tne rniddle two are close enougli to torin a water water nydrogen bond ine constriction region and pseudo two told axis centred abouttne NPA rnotits are indicated by lignt blue and blaclltarrows respectively lne tigure was produced using tntltscttll l36 and RasteralJ s15 articles Figure 5 Resrnues nennrne tne constrlctlon reglon Resrnues rnvolven m tne formation of tne 00m Constrlctlon reersn H102 R197 F58 and Cl lare neprcten rn sons colours wnrle tne transparent slde cnarns are those ottne enuwalent resrnuestounn rn tne structure of SW 191 R206 W48 and F200 lnese slde cnarn dlfferences result n a largerann more hydmphoblc Constrlctlon reglon rn GlpF Alrenrnentsttne resruues was penonnen 051110 a least square fltofmelr backbone atoms lne neure was produced 115mg MOLSCRlPT and RasterBD axis one nearest and hydrogen bonded to the 32 nitrogen ofN194 and the other nearest and hydrogen bonded to nitrogen 32 ofN78 The fourth water visible in the channel is located near the cyto phsmic end ofthe selectivity lter The backbone carbonyl oxygens of residues H76 and A75 form the coordinating hydrogen bonds with this water These four waters do not form a contiguous hydrogen bonded chain as only the middle two are close enough to each other to form a water water hydrogen bond The hydro phobicity pro le of the residues lining the pore indicates that there are three hydrophilic nodes distributed along the length of the selectivity lter Fig 3 As would be expected the four waters identi ed within the sdectivity lter are all located at these nodes Mechanisms of water selectivity Through a combination of channel sterics and solute binding sites AQPI facilitates the rapid and highly selective throughput of water A steric limit of 28 A is established at the constriction region and the chemical properties of the residues forming this structure provide additional criteria for solute selection From the steric limit alone it is now clear why the transport of glycerol by AQP1 is highly unfavourable For a molecule ofwater to diffuse across the narrow AQPI constriction region its effective diameter must be reduced by shedding waters of hydration For this to be an energetically favourable process interactions with primary hydra tion shell waters must be replaced by interactions with residues lining the channel wall over as small a diffusion distance as possible In AQP suf cient hydrogen bond forming groups are avaihble so that water molecules can readily move through the constriction region These bond forming groups are provided by constriction region residues H182 conserved across thewater speci c aquapor ins and R197 conserved throughout most of the aquaporin superfamily and the backbone carbonyl oxygens of residues G190 C191 and G192 In addition to the bound water found at the constriction region there are three additional waters in the selectivity lter Averaging only about 4A in diameter the selectivity lter is also rather hydrophobic but is punctuated by water binding regions at 815 to 2001 Macmillan Magazines Lid several hydrophilic nodes Fig 3 The availability ofwater binding sites at these nodes reduces the energy barrier to water transport across this predominantly hydrophobic pathway while the relatively low number ofsuch sites keeps the degree of solute pore interac tion to a minimum In balancing these opposing factors the aquaporins are able to transport water selectively while optimizing permeability Whereas the aquaporins are optimized for the rapid transport of water some members of the aquaporin family also facilitate the transport of other solutes The bacterial aquaglyceroporin homologue GlpF is optimized for the rapid transport of glycerol Although water has favourable steric accessibility through both the AQP and GlpF channels there is a critical difference in glycerol steric accessibility between these two pores the constriction region of GlpF is almost 1A wider than in AQP1 The hrge difference in glycerol permeability between AQP and GlpF is effectively bridged in aquaglyceroporin AQPS which transports both water and glycerol at moderate rates The hydropathy pro les for the selectivity lter regions ofAQP1AQP3 and GlpF are very similar Most of the amino acid differences occurring in this region are moderate such as swapping one type ofhydrophobic residue for another yet changes to only one or two residues of the constriction regions in these channels are sufficient to radically alter solute selectivity It appears that in AQP1 the residue most critical in supporting rapid water throughput while hindering the passage of glycerol is H182 Fig 5 Throughout the aquaporin family the choice of amino acid at this location appears to be essential in de ning whether an aquaporin will be speci c for water or addi tionally sdective for other solutes such as glycerol In GlpF the replacement ofthis histidine by glycine serves both to signi cantly increase the size of the constriction region and sterically to support a second residue change 3191 to phenylalanine These changes in turn alter the polar nature of this area and result in improved channel interactions with the hydrophobic backbone of glycerol H182ofAQP1isprobablyalso replacedbyglycineinAQP3Aswith GlpF such a substitution should provide the additional room needed to accommodate the side chain of a second probable substitution in this case a tyrosine for C191 In contrast to the substitution for phenylalanine occurring at this position in GlpF the switch to tyrosine potentiallyprovides an additional location for solute hydrogen bonding As a consequence of these two substitu tions H182G C191Y it is expected that the constriction region of AQP3 is similar in size to that of GlpF although somewhat more polar thereby allowing for the moderately rapid transport of glycerol as well as water The amphipathic nature of the selectivity lter has a key role in the rapid transport ofwater in AQP1 and this property is preserved across the superfamily of aquaporins It has been shown to be particularly bene cial for glycerol transport The hydrophobic face ofthe GlpF selectivity lter provides an ideal match for the carbon backbone side of glycerol while the opposing hydrophilic face is equipped with hydrogen bonding groups to replace waters of hydration in the vicinity ofglycerolhydroxyl groups Both glycerol and water molecules were visualized within the GlpF selectivity lter indicating that at high concentrations of glycerol there should be almost one to one transport of glycerol and water Although GlpF permeability to water is signi cant it is substantially less than that measured for its bacterial aquaporin counterpart and AQP1 homologue qu2 ref 22 The constriction region ofGlpF is wider than that ofAQPl so it would appear that the more hydrophobic nature of GlpF is the primary factor responsible for its relatively reduced ability to transport water Preselection of solutes by the vestibules may also be important in maximizing rates of transport The relatively stronger hydrophobic nature of the vestibules in GlpF should improve the transport rates of solutes that contain a signi cant hydrophobic component such as glycerol although potentially at the expense of rapid water NAmulvOL an my DECEMBERznm lwwwnannecom throughput Conversely the more hydrophilic vestibules of AQPl favour the preselection of water Barriers to ion transport In the aquaporin and KcsA K r channel selectivity lters carbonyl groups extending from inner helix linker loops face the pore staggered along the channel axis In AQPl and as reported in the structure of GlpF these ten pore accessible carbonyl oxygen atoms are distributed along a 25 A stretch along one side of the pore For KcsA this zone is signi cantly shorter con ned to half of the transmembrane pore and yet contains 16 carbonyl oxygen atoms arranged in a stacked ring con guration with four carbonyls per ring hydrated K4r ions are closely and symmetrically surrounded by carbonyl oxygen atoms so that making coordination site transfers between hydrating water and the carbonyl oxygens is energetically favourable Hydrated ions encountering the type of selectivity lter utilized by the aquaporins would nd that the available carbonyl groups and water coordinating residues such as R197 and H182 in AQP1 could only effectively substitute for less than half of the hydrating waters at any point along this segment of the channel Such partially hydrated ions would still be too large to pass through the constriction region or most of the selectivity lter Positively charged ions would nd additional resistance to their transport from repulsive forces generated through interactions with the dipoles of helices M3 and M7 oriented with their positive ends pointed into the pore the highly conserved R197 located near the constriction region the amines of N78 and N194 located about the pseudo two fold axis and by the histidines H182 and H76 located at opposite ends of the selectivity lter Negatively charged ions would experience repulsive forces from the many pore accessible carbonyl oxygen atoms lining the selectivity lter and inner regions of the vestibules Water networks or chains have been seen in the high resolution structures of proteins such as the photosynthetic reaction centre23 and cytochrome b5fref 24 and it has been suggested that protons could be transported along a continuous linear network of suitably oriented hydrogen bonded waters proton wire by a transfer process termed the Grotthuss mechanism25 25 In this mechanism a proton can be instantaneously shuttled along this special network of water molecules However no suitable chain of hydrogen bonded water molecules spanning the selectivity lter has been located in the density map of the AQPl crystal structure Factors preventing the establishment of such a hydrogen bonded water chain along the string of carbonyl oxygen atoms lining the AQPl channel include the following highly conserved elements arginine located at the extracellular side of the selectivity lter the two asparagines and helical dipole end charges located in the pore about the pseudo two fold axis and the histidines located at the ends of the selectivity lter The two positive helical dipole end charges are by themselves suf cient to disrupt any suitably oriented alignment of water dipoles along the channel pore D Methods Crystallization of AOP1 Native crystals were obtained as described previously Soaking of crystals in a variety of heavycatom compounds greatly affected diffraction quality even for very low concentra tions of heavy atom compounds Coccrystallization yielded thaliumcderivatized crystals suitable for providing initial phasing information Data collection and processing MAD four wavdength data sets were collected from TchQPl crystals at the Advanced Light Source ALS of Lawrence Berkeley National Laboratory Diffraction data were processed with DENZOISCALEPACK Crystals belong to space group 1422 unit cell dimensions are a l 933 c 1805 A d I y 90 and contain one molecule per asymmetric unit the best crystals diffracted to 21 A A Patterson map based on dispersive and anomalous differences showed strong heavycatom pealcs located near the crystals fourcfold symmetry axis these positions were re ned with the use ofthe SHARR program The re ned heavycatom positions were used to obtain an initial density map NATURE lVOL 414 l 2027 DECEMBER 2001 lwwwnaturecom 2001 Macmillan Magazines Ltd articles An improved density map was produced withthe application ofsolvent flatteningthrough the program DM and was used to build an initial model at 28A resolution Model building Modelbuildingwas performed using program 03 while model re nementwas conducted using the Crystallographic and NMR system CNS Extension ofphases and model re nement to 22 A was accomplished with a data set obtained from crystals grown in the presence ofgold cyanide but for which no multicwavelength anomalous differences were observed Others have also obtained improvements in crystal order using gold cyanide as a crystallization additive The diffraction data from these crystals extended to 21 A but were signi cantly anisotropic which resulted in a reduction in the percentage of statistically reliable data in the direction ofthe hek plane CNScbased re nement ofthe model residues 1449 ofa possible 271 using data to 22A resolution resulted in an improved model with an R7 value of308 and a crystallographic R of266 The zFo Fc electron density map obtained from these data is however of good quality Fig 1d the relatively high values ofR and R7 are a consequence ofthe diffraction set anisotropy such an effect was expected and larger R factors have been reported for anisotropic data sets of similar resolution which also yielded good quality maps as reported in ref 34 Received 11 July accepted 30 October 2001 l lshibashi K or al Cloning and functional expression of a new aquaponn AQP9 abundantly expressed in the peripheral leukocytes permeable to water and urea but not to glycerol Btochem Braphys RES Commun 244 268474 1998 2 Borgma M or al Cellular and molecular biology of the aquaponn water channels Army Rov Btochem 68 425458 1999 3 Meinild A K oral Bidirectional water uxes and speci city for small hydrophilic molecules in aquaponns 0e5I Biol cliom 273 32445e32451 1998 4 Verkman A 5 Water channels in cell membranesAmia Rov PhySml 54 97408 1992 5 Agre P oral Aquaponn CHIP the archetypal molecular water channel Am I PhySml 265 F463e e475 1993 5 Denker e M or al identi cation puri cation and partial characterization of a novel M 28000 integralmembrane protein from erythrocytes and renal tubules I Biol Chem 2631553445542 1988 7 Zeidel M L or al Ultrastructure pharmacologic inhibition and transport selectiVity of aquaponn channelcformmg integral protein in proteoliposomes Biochomisny 33 15054515 1994 8 lap e K 8r Li HcL structure of osmocregulated H10 channel AQPcCHIP in prolecuon at 35A resolutionI Mal Btol 251413e420 1995 9 Li HcL Lee 5 8r lap e K Molecular design of aquaponncl water channel as revealed by electron crystallography Nature Snacr B101 4 253e255 1997 10 Cheng A or al threedimensional organization of a human water channel Nature 337 527e530 1997 ll Walz T or al the threedimensional structure of aquaponncl Name 337 524e527 1997 12 Ren G or al threedimensional fold of the human AQPI water channel determined at 4 A resolution by electron crystallography of twocdlmensmnal crystals embedded in ice I Mal B101 301 3694387 2000 13 Murata K oral structural determinants ofwaterpermeationthrough aquaponnc 1 Nature 407 599e 505 2000 14 Ren G or al visualization of a Watercselectwe pore by electron crystallography in Vttreous ice Pro NarlAcarl Sci USA 9313984403 2001 15 Fu D or al structure of a glycerolcconductmg channel and the basis for its selectiVity Science 290 481486 2000 15 smith e L 8r Agre P Erythrocyte M 28000 transmembrane protein exists as a multisubunit oligomer similarto channel proteinsI Btol cliom 266 5407e5415 1991 17 Nielsen s oral Distribution ofdteaquaponn CHIP in secretory and resoiptive epithelia and capillary endothelia Proc Natl Amd Sci USA 90 72754279 1993 18 Werner s l oral Anewforce eld for molecularmechan articles 31 Jones T A er a1 improved methods for building protein models in electron density maps and the location of errors in these modelsActa nymzllagv A 47 110419 1991 32 Brunger A T er a1 Crystallography amp NMR system A new software suite for macromolecular structure determinationAcra Crymzllagv D 54 9054221 1998 33 chang G er a1 Structure ofd39le MseL homo1og from Mycoliacrerinm nibmnzom a gated medlane osensitive ion channel Science 232 zzzuezzze 1998 34 Groll M er al The catalytic sites onDS proteasomes and their role in subunltmaturatlon a mutational and crystallographic study Pm NarlAcad Sci USA96109767109831999 3s Ihompson 1 D er a1 CLUSTAL w improying the sensitiyity of progressive multiple sequence alignment through sequence weighting positionrspeu r gap penalties and weight matrut choice NnczocAcitis Re 22 45734580 1994 Kraulis p 1 MOLSCRIPT a program to produce both detailed and schematic p1ots ofpmteln structures Appz Crymzllogv 24 9454250 1991 Merritt EA amp Baton D 1 Raster3D photorealistiemoleeular graphics Morhodslsnzymoz 277 suse 524 1997 Smart 0 s oral Thepore dimensions Ofgramicidin A Biophys I 65 245572450 1993 Kyte1 ampDoolltdeR F A simplemerhodtor displaying thehydropathie character ofapmteln 1 Mol Biol 157 105432 1982 3 3 3 3 878 2001 Macmillan Magazines Ltd Acknowledgements Preliminary data collection and screening for heavyeatom derivatives were conducted at beamlines X25 at the National Synchrotron Light Source and 175 at Stanford Synchrotron Radiation Laboratory data sets used in determining the modelwere collected at Beamline 502 Advanced Light Source Lawrence Berkeley National Laboratory We would like to thank all staff members ofthese beamlines for their assistance and Brc Wang for discussions on heavyeatom position re nement This work is supported by funding from the National Institutes of Health and by the Of ce of Health and Environmental Research US Department ofEnergy The coordinates have been deposited in the Protein Data Bank under accession number HAN Competing interests statement The authors declare that they have no competing nancial interests Correspondence and requests for materials should be addressed to BKI email BKIap1b1gov or 12w email PIWalian1b1gov NATURE VOL414 2027 DECEMBER 2001 wwwnaturecom
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