1TM8 =>superSeed =>1YMG1YMGMar The structure starts with residue 6 Ser and ends with residue 239 Glyrepresenting 88% of the molecule. 10 water molecules atoms of HOH oxygen H2O Five residues at the N terminus and 24 residues at the C terminus are not well ordered in the structure. H1,H2,H3,H4,H5,H6, C-terminal helix H7,H8 and the five loops are named A, B, C, D, E . The NPA boxes are located in the loops LB and LE, which are rather hydrophobic in nature and have short (half) helices HB and HE and loops named LB,LE off .
An apparent paradox is that the lens fiber cell must keep its interior relatively dry to maintain crystallin protein transparency, but it invests a significant percentage of its cellular resources to produce large amounts of AQP0, a member of the water channel family. Why should the fiber cell invest so much synthetic energy to produce a protein that could seemingly compromise crystallin transparency? Clearly, the lens requires a basal level of water conductance for good health and the great longevity of the lens cells, most of which live for the entire lifespan of the organism. However, the fiber cell compensates for having an incredibly large number of these channels (˜60% by weight of all membrane proteins in the fiber-cell plasma membrane is AQP0) by having AQP0 conduct water very poorly. Thus, it ensures a uniform response to osmotic challenge in all areas of the cell surfaces of the tightly packed fiber cells throughout the lens and maintains homogeneous transparency throughout the lens. Measurements of water conductance using oocyte and proteopositivelysome swelling demonstrate that AQP0 water permeability is 15- to 45-fold less than AQP1. Published water-permeability data have varied from 0- to 43-fold over conduction through lipids alone or through the membranes of oocytes injected with water. Unfortunately, comparisons between published conduction rates are difficult because they are generally relative conductances uncorrected for the number of conducting channels, and they are also difficult because of the variety of materials and methods used. A question arises as to the channel dimensions required for passage of various permeants through the channel. Use of the minimum diameter of the permeant as a rough measure of the channel diameter required for passage, as well as the diameter of the largest sphere that will fit in the channel at the narrowest constriction of the channel, provides one criterion. The diameter of the channel calculated in this way for a static structure would suggest that both of our structures of AQP0 channel (d = 1.5 Å) and the Walz structures of AQP0 channel (d = 2.0 Å) are too narrow to permit the passage of water and other larger permeants, including glycerol and urea. Previous functional studies have shown significant measurable flux through AQP0 of all three of these substances, even though some of these results are questionable. However, if the channel were to have a noncircular profile, then the available cross-sectional area could be larger than the value implied by this calculation. Further, the channel diameter values calculated for bAQP0, AQP1, and AQPZ are also all smaller than the accepted value of 2.8 Å for the diameter of a single water molecule H2O, yet all of these AQPs conduct water at close to the diffusion-limiting rate. Therefore, to test the possible accommodation of AQP0 to these substrates, we selected side-chain rotamers of constriction-region residues of our AQP0 structure that maximized channel diameter without any main chain movement. After extensive energy minimization and annealing, the resulting structures had stable rotamers that could enlarge the channel diameter to slightly >2.9 Å, which is more than large enough for water to pass. Additional circumstantial evidence of water transport is the presence of eight Helix-bonded water 8 H2O molecules in the channel (no waters are seen in the electron-diffraction structure). These waters are moderately well ordered, as reflected by their electron densities (Graph Center) and by their B factors, which are close to the average for the protein (?B? = 55) as follows: 57,57,54,51,48, 44,41, and 38, from extracellular to intracellular in the channel. Thus, there is water throughout the channel pathway (Graph). The two channel-constricting tyrosines (Tyr23 and Tyr149) that are totally conserved in all known AQP0s, suggests the possibility of “kinetic limiting” (namely, if both tyrosines have to move out of the channel simultaneously to achieve high conductance, the probability of both of them allowing passage together is the product of the probabilities of each one individually).
1YMG H1,H2,H3,H4,H5,H6,H7,H8,A,B,C,D,E,HB,HE,LB,LE 10 water molecules atoms of HOH oxygen off H2O Backbone thin off Modulation by changes in pH or ion concentration have been suggested in the control of transport of water and possibly other substrates O2, NO,CO through AQP0. His-40 has been suggested to be the residue off that is responsible for the inhibitory effect of pH or Ca2+ on water transport rates. Therefore, Nemeth-Cahalan and Hall made mutations that changed His-40 to alanine, aspartate, or lysine, and they showed that treatment of oocytes expressing the mutants no longer displayed the pH<6.5-dependent closing as acidity was raised to pH<6.5. Reaction of histidines in AQP0 with diethylpyrocarbonate (DEPC) removed pH dependence and actually increased water conductance (restored by histidine-specific reversal with hydroxylamine), adding support to the case that titration of histidines alters conductance. Comparisons of the x-ray and electron crystal structures. View down the monomer channel z axis from the extracellular side showing the positions of Tyr23 and Tyr149 residue off can see the positions of the three histidines that are close to the channel and vestibules. His40,His66 and His172 residue off VI. Posttranslational Modification Cytosole site M8 helix 2B6PMarzH1,H2,H3,H4,H5,H6,H7,A,B,C,D,E,HB,HE,LB,LE 10 water molecules atoms of HOH oxygen off H2O HOH360,264,270,272,279,280,308,314,316,392 1YMG H1,H2,H3,H4,H5,H6,H7,H8,A,B,C,D,E,HB,HE,LB,LE 10 water molecules atoms of HOH oxygen off H2O Backbone thin off Posttranslational modifications have also been suggested in the regulation of AQP0 activity. The lenses were taken from 1- to 2-year-old cattle in good health, so most age-related covalent additions, such as glycosylation and phosphorylation to 1YMG.pdb, were minimized. Ser235 (Ser229, Ser231are), Ser243, and Ser245 residues off that in the C-terminal chain have been implicated as regulatory phosphorylation sites in bAQP0. 1YMG.pdb not shown Ser243, and Ser245. In extracellular westibule are shown : Ser31,Thr108,Ser126,Thr120,Thr199,Thr195residues of OH groups contaning residues off 6 OH sites for phosphorylation. Two of Ser243, and Ser245 residues are in the mobile helix M8 C-terminal 24 amino acids (239–263) in 2B6P.pdb are present in our structure of 2B6P.pdb. Push on the molecule by right baton of mouse and choose: 1. Select>Residue>SER and second time push on the molecule choosing 2. Display>Sticks and Color>CPK. VII. Conformational Consequences of Junction Formation View down the monomer channel z axis from the extracellular side showing the positions of the three histidines that are close to the channel and vestibules. (Lower Right) Side view. The extracellular hydrophobic residues involved in cell-to-cell contacts are highlighted and labeled Leu39,Pro38,Gly37,Ala35 Gly114,Val112,Ala111,Pro110,Pro109; Val125,Gly124,Pro123,Leu121; Phe198,Leu194 residues off not shown Pro36. In the comparison junctional formation does not produce any closure per se. Junction formation does not appear to lead to any physical closure of the channel. Walz and coworkers showed that loop C-terminus helix M8 mediates most of the junction-forming interactions. Some Suggest that it may be altered by the contact formation, suggesting that if this possibility were true, it could stabilize an alternate conformation of Arg-187 off located in the central “selectivity filter SF” of the channel to contribute to channel closure. Arg187 makes two good Helix bonds from NH1 to the C=O of Pro191 and Arg187 NH1 to C=O of Ala-117, which we find to be conserved throughout all AQPs. Because of a slight difference in placement of Arg187, the electron-diffraction structure claims a Helix-bonded contact between the amidinium cation of Arg187 and Asn119; however, the geometry of this interaction is 90° from ideal and seems to be unlikely. Improbable is that close approximation of adjacent extracellular domains acts as a gating mechanism by inducing distortion of the channel in this region either. Regarding whether the close apposition of AQP0 tetramers could form aligned, sealed cell–cell junctions, one asks whether the very close approximation of the extracellular domains of AQP0 tetramers closes off access to the channels from the extracellular environment. We answer no; there are several large gaps between the opposing extracellular domains of AQP0 in both the electron-diffraction structure and in a structure in which the x-ray structure is overlaid on the double-membrane configuration. These portals are large enough to allow the access of intercellular water molecules to the extracellular vestibule of all channels. VIII. Charged amino acid side chains in the extracellular surface 1YMG H1,H2,H3,H4,H5,H6,H7,H8,A,B,C,D,E,HB,HE,LB,LE 10 water molecules atoms of HOH oxygen off H2O Backbone thin off His40,Arg33,Arg113,His122,Arg196 Counting charged amino acid residues off side chains in the extracellular surface of the bAQP0 shows the surface to be net +5 positively charged at physiological pH=7.36. This observation helps to explain the observed association between the extracellular domain of AQP0. With each opposing surface having a net positive charge +5, would tend to repel each other without the hydrophobic interactions. When hydrophobic interaction dominating the other type of junctional AQP0–AQP0 contacts are stronger. Leu39,Pro38,Gly37,Ala35 Gly114,Val112,Ala111,Pro110,Pro109; Val125,Gly124,Pro123,Leu121; Phe198,Leu194 residues off not shown Pro36. IX. Aquaporin-0 membrane junctions of a closed water pore 1.1SORMarz His40,Arg33,Arg113,His122,Arg196 Countainig charged amino acid residues off side chains 2.Hydroxyl groups 6 OH sites for phosphorylation. Ser31,Thr108,Ser126,Thr120,Thr199,Thr195 OH groups contaning residues off . 3.Conformation of Arg-187,Asn68 off located in the central “selectivity filter SF” of the channel to contribute to channel closure. Arg-187 makes two good Hydrogen bonds from NH1 to the C=O of Pro191,Phe189,Asn184, Arg187,Ser31 NH1 to C=O of Asn115,Asn115,Ala-117,Asn119 close the water pore Val41,Tyr177 which we find to be conserved throughout all AQPs. 4.hydrophobic residues involved in cell-to-cell contacts are highlighted and labeled Leu39,Pro38,Gly37,Ala35 Gly114,Val112,Ala111,Pro110,Pro109; Val125,Gly124,Pro123,Leu121; Phe198,Leu194 residues off not shown Pro36. Hydrophobic interactions that we see dominating the other type of junctional AQP0–AQP0 contacts. X. membrane aquaporin-0 protein interactions with annular lipids half closed 2B6O.pdb and open 2B6P.pdb water channel 1.2B6OMarzH1,H2,H3,H4,H5,H6,H7,A,B,C,D,E,HB,HE,LB,LE 5 water molecules atoms of HOH oxygen off H2O HOH274,HOH275,HOH276,HOH293,HOH314 Determined the 2.5 Å structure of AQP0 in two-dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Hydrophobic - non polar amino acids side chains bind phosphatidylcholine (DMPC) lipid hydrocarbon chains by van der Walls forces (London forces) on each contact point -2 kJ/mol. 1)Arg196,Ala102,Val103,Tyr105,Ser106,HOH291,HOH301,HOH327,Leu83,Leu84,Ile87,Val90,Val91,Leu94,Lys238 residues off DMPC,269,270,272,291,267,271 lipids off 2)Arg5,Ser6,Phe9,Trp10,Leu84,Cys88,Ala7,Arg11,Phe14 residues off DMPC,266,272,264 lipids off 3)Leu95,Tyr105,Ile193,Leu194,Arg196,HOH326 residues off DMPC,266,265,264,267,269 lipids off Backbone thin off References 1.(2004) Proc.Natl.Acad.Sci.USA 101: 14045-14050 1TM8 =>superSeed =>1YMG 2. Nature. 2004 May 13;429(6988):193-7.1SOR 3.EMBO J. 2010 May 19; 29(10): 1652–1658. 3M9I 2B6O Back to the Index...
1YMG H1,H2,H3,H4,H5,H6,H7,H8,A,B,C,D,E,HB,HE,LB,LE 10 water molecules atoms of HOH oxygen off H2O Backbone thin off Posttranslational modifications have also been suggested in the regulation of AQP0 activity. The lenses were taken from 1- to 2-year-old cattle in good health, so most age-related covalent additions, such as glycosylation and phosphorylation to 1YMG.pdb, were minimized. Ser235 (Ser229, Ser231are), Ser243, and Ser245 residues off that in the C-terminal chain have been implicated as regulatory phosphorylation sites in bAQP0. 1YMG.pdb not shown Ser243, and Ser245. In extracellular westibule are shown : Ser31,Thr108,Ser126,Thr120,Thr199,Thr195residues of OH groups contaning residues off 6 OH sites for phosphorylation. Two of Ser243, and Ser245 residues are in the mobile helix M8 C-terminal 24 amino acids (239–263) in 2B6P.pdb are present in our structure of 2B6P.pdb. Push on the molecule by right baton of mouse and choose: 1. Select>Residue>SER and second time push on the molecule choosing 2. Display>Sticks and Color>CPK.
View down the monomer channel z axis from the extracellular side showing the positions of the three histidines that are close to the channel and vestibules. (Lower Right) Side view. The extracellular hydrophobic residues involved in cell-to-cell contacts are highlighted and labeled Leu39,Pro38,Gly37,Ala35 Gly114,Val112,Ala111,Pro110,Pro109; Val125,Gly124,Pro123,Leu121; Phe198,Leu194 residues off not shown Pro36. In the comparison junctional formation does not produce any closure per se. Junction formation does not appear to lead to any physical closure of the channel. Walz and coworkers showed that loop C-terminus helix M8 mediates most of the junction-forming interactions. Some Suggest that it may be altered by the contact formation, suggesting that if this possibility were true, it could stabilize an alternate conformation of Arg-187 off located in the central “selectivity filter SF” of the channel to contribute to channel closure. Arg187 makes two good Helix bonds from NH1 to the C=O of Pro191 and Arg187 NH1 to C=O of Ala-117, which we find to be conserved throughout all AQPs. Because of a slight difference in placement of Arg187, the electron-diffraction structure claims a Helix-bonded contact between the amidinium cation of Arg187 and Asn119; however, the geometry of this interaction is 90° from ideal and seems to be unlikely. Improbable is that close approximation of adjacent extracellular domains acts as a gating mechanism by inducing distortion of the channel in this region either. Regarding whether the close apposition of AQP0 tetramers could form aligned, sealed cell–cell junctions, one asks whether the very close approximation of the extracellular domains of AQP0 tetramers closes off access to the channels from the extracellular environment. We answer no; there are several large gaps between the opposing extracellular domains of AQP0 in both the electron-diffraction structure and in a structure in which the x-ray structure is overlaid on the double-membrane configuration. These portals are large enough to allow the access of intercellular water molecules to the extracellular vestibule of all channels.
1.2B6OMarzH1,H2,H3,H4,H5,H6,H7,A,B,C,D,E,HB,HE,LB,LE 5 water molecules atoms of HOH oxygen off H2O HOH274,HOH275,HOH276,HOH293,HOH314 Determined the 2.5 Å structure of AQP0 in two-dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Hydrophobic - non polar amino acids side chains bind phosphatidylcholine (DMPC) lipid hydrocarbon chains by van der Walls forces (London forces) on each contact point -2 kJ/mol. 1)Arg196,Ala102,Val103,Tyr105,Ser106,HOH291,HOH301,HOH327,Leu83,Leu84,Ile87,Val90,Val91,Leu94,Lys238 residues off DMPC,269,270,272,291,267,271 lipids off 2)Arg5,Ser6,Phe9,Trp10,Leu84,Cys88,Ala7,Arg11,Phe14 residues off DMPC,266,272,264 lipids off 3)Leu95,Tyr105,Ile193,Leu194,Arg196,HOH326 residues off DMPC,266,265,264,267,269 lipids off Backbone thin off