Báo cáo Y học: Modeling the three-dimensional structure of H+-ATPase of Neurospora crassa Proposal for a proton pathway from the analysis of internal cavities pptx

Eur J Biochem 269, 5246–5258 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03236.x Modeling the three-dimensional structure of H+-ATPase of Neurospora crassa Proposal for a proton pathway from the analysis of internal cavities Olivier Radresa1, Koji Ogata2, Shoshana Wodak2, Jean-Marie Ruysschaert1 and Erik Goormaghtigh1 Service de Structure et Fonction des Membranes Biologiques, Universite´ Libre de Bruxelles, Bruxelles, Belgium; 2Unite´ de Conformation des Macromole´cules Biologiques, Universite´ Libre de Bruxelles, Bruxelles, Belgium Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as ˚ template the 2.6 A crystal structure of rabbit sarcoplasmic 2+ reticulum Ca -ATPase [Toyoshima, C., Nakasako, M., Nomura, H & Ogawa, H (2000) Nature 405, 647–655] Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue The 3D structures have been determined for only a limited number of membrane proteins Growing large, well ordered, 2D or 3D crystals of membrane proteins remains indeed a major limiting step for X-ray or electron crystallography Alternative approaches for obtaining structural information are therefore very useful One such approach is the homology modeling technique whereby a known 3D structure of a related protein is used as a template for building an atomic model from the amino acid sequence of the target protein Although validity of the resulting model requires experimental confirmation, it can provide useful insights into the structure–function relationship in related enzymes The plasma membrane H+-ATPase of the fungi Neurospora crassa (referenced hereafter as PMA1_NEUCR) is a member of the large and ubiquitous P-type ATPase family This family currently counts almost 200 members involved in the transport of a variety of ionic substrates including charged amino phospholipids [1] PMA1_NEUCR comprises a cytoplasmic catalytic site responsible for MgATP hydrolysis that is anchored in the membrane by 10 transmembrane segments As other P-type ATPases, PMA1_NEUCR is fully active as a monomeric 100 kDa polypeptide chain, it transports ions outside the cell in an electrogenic way using energy from MgATP hydrolysis and its catalytic cycle is characterized by the formation of a covalent enzyme-aspartyl phosphate intermediate [2,3,42,43] The 3D structures of PMA1_NEUCR and of another P-type ATPase, the Ca2+-ATPase of rabbit sarcoplasmic reticulum (referenced hereafter as ATC1_RABIT), have ˚ been determined at A resolution [4,5] At this resolution, the electron density map is accurate enough to depict the packing and tilt angle of each of the transmembrane segments Comparison of the two low-resolution models, which are believed to represent different conformational states, revealed that they displayed strikingly similar packing of their respective 10 transmembrane segments whereas their cytoplasmic domains appeared too different to allow direct superimposition [6,7] Recently, the resolution of the structure of ATC1_ ˚ RABIT was increased to 2.6 A providing the first structure at near atomic resolution for a P-type ATPase [8] This latter ´ Correspondence to E Goormaghtigh, Universite Libre de Bruxelles, Campus Plaine CP 206/2, B 1050, Bruxelles, Belgium Fax: +32 26505382, Tel.: +32 26505386, E-mail: egoor@ulb.ac.be Abbreviations: HAD, haloacid dehalogenase; TM, M, transmembrane segment; PSP, phosphoserine phosphatase Enzymes: PMA1_NEUCR, Neurospora crassa plasma-membrane H+-ATPase; ATC1_RABIT, Oryctolagus cuniculus (rabbit) Ca2+ATPase of sarcoplasmic reticulum (splice isoform SERCA1a) (Received 27 May 2002, revised 23 August 2002, accepted September 2002) Keywords: neurospora; P-ATPase; homology model; cavity; H+ 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5247 Ó FEBS 2002 structure obtained in presence of two buried calcium ions is believed to represent an open conformational state analog˚ ous to the previously determined A resolution PMA1_NEUCR structure This, together with the striking similarities between the ˚ ˚ A electron density maps, suggests that the 2.6 A structure of ATC1_RABIT would be a valid template for building an atomic model of PMA1_NEUCR Recently, partial models comprising the first six transmembrane segments and a portion of the cytoplasmic loop responsible for ATP hydrolysis were built for plant and yeast H+-ATPases on the basis of the ACT1_RABIT crystal structure [9] From these models is was proposed that proton transport in the H+-ATPases is mediated through specific binding of a hydronium ion at a site structurally equivalent to the second calcium binding site in ACT1_RABIT In this work, we combine homology modeling techniques and transmembrane topology predictions to build a model of PMA1_NEUCR that comprises all 10 transmembrane helices This model is then used to propose an alternative hypothesis for the proton transport pathway This hypothesis is based on the assumption that as in the case of the well-known bacteriorhodopsin proton pump, H+ ions would be the relevant transported species, with their transport mediated by one or more acidic side chains of the protein and a network of interacting buried or partially buried water molecules [10] To find pathways consistent with this hypothesis, the PMA1_NEUCR model is used to compute the positions of internal polar cavities that are large enough to contain at least one water molecule Analyses of X-ray structures of soluble proteins have indeed shown that such cavities usually harbor bound water molecules [11–13] Furthermore, control calculations reported here, in which the same approach is applied to the highest resolution structures of the proton pump bacteriorhodopsin and halorhodopsin, reveal a good correspondence between the positions of identified polar cavities, water molecules and residues believed to mediate proton transport in the proton pump Calculations performed on our H+-ATPase model identify a string of internal polar cavities, tracing a welldefined pathway connecting the phosphorylation site in the extracellular domain to the intracellular side of the molecule Most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis Some are also in contact with residues whose role in proton transport has as yet not been analyzed This pertains in particular to residues in the cytoplasmic domain which might be involved in the pathway of proton entry In the current absence of detailed 3D data, these suggestions could be tested by mutatagensis experiments Furthermore, the approach might be a useful preliminary tool for identifying putative proton pathways in other proton-transporting enzymes for which structural data are available MATERIALS AND METHODS Building the 3D model of PMA1_NEUCR In our approach, the model of PMA1_NEUCR was built using the ATC1_RABIT crystal structure as template by combining transmembrane topology predictions with standard homology modeling techniques This was necessary as sequence similarity between the template and target proteins is rather low for much of their C-terminal portion Transmembrane topology predictions The transmembrane topology of PMA1_NEUCR was predicted from the amino acid sequence by averaging the results of five different predictive algorithms: DAS [14], PHDHTM [15], HMMTOP [16], TMHMM [17] and TMPRED [18] To assess the accuracy of the predictions, the same algorithms were applied to the ATC1_RABIT protein and the results were compared with the topology defined in the ˚ 2.6 A structure (1EUL.pdb) (Table 1), taking into account the position of aromatic residues at the boundaries of the transmembrane segments It was verified that none of these algorithms included this 3D structure in its learning set This is certainly important for the DAS and TMPRED algorithms which rely directly on a database of known structures The predictions made for PMA1_NEUCR were compared with experimental data on the insertion into microsomes of recombinant peptides from helices M3, M5, M7, M8 and M10 [19,20] (Table 2) In the case of ATC1_RABIT, combining the predictions from the different algorithms yielded accurate predictions for nine out of 10 transmembrane segments This is consistent with previous reports where it has been shown that secondary structure predictions tend to be improved upon averaging the results from different methods [21] The average transmembrane topology for PMA1_NEU CR, presented in Table 2, contains 10 transmembrane Table Topological predictions and average topological model for ATC1_RABIT Comparison with the topology of the crystal structure DAS M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 (1.7) 60–76 88–104 – 263–276 294–308 769–784 – 832–851 – 930–954 967–980 HMMTOP PHDHTM TMHMM TMPRED Average 60–78 85–106 207–228 261–279 286–307 772–793 – 838–859 – – 967–980 59–76 83–106 – 259–276 295–314 773–794 – 833–850 – 932–949 – 60–78 85–107 – – – – – 837–859 – 928–950 – 60–78 85–107 207–226 260–279 292–314 769–790 – 839–855 896–917 928–950 – 60–77 85–106 – 261–278 292–311 771–790 – 835–855 896–917 930–951 967–980 57–77 88–107 – 256–279 288–312 760–778 789–809 832–854 891–914 932–952 967–986 Ó FEBS 2002 5248 O Radresa et al (Eur J Biochem 269) Table Topological predictions and average topological model for PMA1_NEUCR Comparison with available data on recombinant peptides Predictive algorithms DAS (1.7) M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 HMMTOP PHDhtm TMHMM TMPRED Average Recombinant peptides 116–126 144–159 291–312 322–348 664–674 691–714 717–734 757–774 – 828–843 854–877 113–137 145–169 289–313 327–351 – 689–713 721–741 755–779 – 822–846 854–878 117–134 142–159 297–314 325–343 – 697–721 – 758–775 – 826–843 859–876 – – 292–314 327–349 – 691–713 720–738 757–775 796–814 829–849 860–878 116–136 141–160 292–310 327–354 660–678 688–713 721–738 755–774 – 827–847 860–877 116–133 143–162 292–313 326–349 – 691–715 720–738 756–775 796–814 826–846 856–878 – – 292–314 – – 688–713 721–738 755–779 807–826 827–847 854–878 ˚ segments in agreement with the A electron density map and with experimental data on recombinant peptides It is worth noting at this point that these data not provide information on the precise boundaries of transmembrane segments but rather on the regions initiating or impairing insertion of a segment in the membrane Therefore, in the case of M8 where the length of the M8 recombinant peptide is probably shorter than that of the M8 helix in the native protein [20], the TMHMM predictions for M8 were used Sequence alignment The sequences of Neurospora crassa H+-ATPase (PMA1_NEUCR) and rabbit SERCA1a/ Ca2+-ATPase (ATCI_RABIT) were retrieved from the Swissprot database [22] Obtaining a correct sequence alignment is the cornerstone of success in all homology modeling procedures Here two different algorithms were used to align the two sequences CLUSTALW [23] was used to generate a global alignment This alignment showed 21.6% amino acid identity In addition the SIM algorithm [24], was used to generate local alignments, where short segments of both sequences were optimally aligned The results from the local alignment obtained with SIM were used to manually adjust the global alignment at the boundaries of the gapped segments In both alignment methods, the Blosum62 substitution matrix was used and the open gap and extension penalties were 12– for CLUSTALW and 12–4 for SIM PHDsec [25] was used to generate secondary structure prediction for PMA1_NEUCR sequence and the DSSP algorithm [63] was used to produce secondary structure assignments from the coordinates of the template The domain displaying the highest sequence similarity between the target and template proteins extends from the N-terminus of ATC1_RABIT up to transmembrane helix M6 This domain includes the large cytoplasmic loop containing the active site responsible for MgATP hydrolysis which is located between M4 and M5 This cytoplasmic loop seems to be shorter in PMA1_NEUCR than in ATC1_RABIT In order to superimpose the strictly conserved ÔVKGAP777Õ and ÔDPPR537Õ motives, ClustalW introduces three large gaps in the PMA1_NEUCR sequence, representing deletions of 14, 17 and 45 residues in ATC1_RABIT The corresponding parts of the 3D model were, however, close enough to be linked by short connecting loops using the loop modeling procedure, as described below These loops are located in the periphery of the structure and not interfere with the core domain Aside from this gapped cytoplasmic loop, sequence identity in this domain exceeded 30% making this part of the alignment more straightforward We could furthermore verify that this sequence alignment was consistent with the alignment between the secondary structures predicted solely from the sequence of PMA1_NEUCR and those assigned in the equivalent regions of the ATC1_RABIT template (Fig 1) The C-terminal domain of both enzymes displayed a much lower level of sequence similarity, a common feature in eukaryotic P-type ATPases [26] In addition, several lines of evidences suggest that PMA1_NEUCR contains an additional cytosolic regulatory domain at the C-terminus This domain is located following the last transmembrane segment M10 Consequently, the raw global alignment produced by CLUSTALW for the C-terminal domain had to be revised, but without the help from local alignments produced by SIM for this region, as those concerned very short segments separated by large gaps Information on the predicted transmembrane topology was therefore used as a guide to align the sequence from M6 onwards Despite the low level of sequence identity, this topology-based alignment was consistent with the CLUSTALW alignment up to segment M7, suggesting that the loops connecting, respectively, M5 and M6, and M6 and M7, would be equally short in both enzymes The region encompassing M8, M9 and M10 were aligned manually by aligning the corresponding transmembrane segments of both enzymes while maximizing sequence identity As mentioned above, the prediction for the M8 segment is consistent with available results on recombinant peptides The position of M10 is consistent with results on the tryptic cleavage of the additional C-terminal regulatory domain in PMA1_NEUCR, which showed that the 897–920 segment had a cytoplasmic location [27] In plant H+-ATPases, where an equivalent domain is also located after M10, this domain was suggested to interact directly with the enzyme active site [28] In the ATC1_RABIT template, the active site is located in the large cytoplasmic loop between M4 and ˚ M5, almost 45 A away from the end of M10 These considerations impose some constraints on the Ó FEBS 2002 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5249 Fig Sequence alignment of PMA1_NEUCR and ATC1_RABIT PMA1_NEUCR PHD: secondary structure prediction of PMA1_NEUCR using the PHDsec algorithm PMA1_NEUCR DSSP: secondary structure of the model structure assigned with the DSSP algorithm (arrows: b-sheets; helices: a-helices) Shaded colored box: transmembrane domains of ATC1_RABIT and PMA1_NEUCR Shaded orange box: PMA1_NEUCR transmembrane segments built with the topology-based alignment Numbers below the sequences refer to the residues in direct contact with the cavities to in PMA1_NEUCR; italic font is used when a mutation has been reported Residues appearing in white over a red background are identical Residues appearing in red over a white background are homologous PMA1_NEUCR model in this region In particular, the regulatory domain of PMA1_NEUCR should be able to reach across the active site If we believe that it adopts an a-helical structure as indicated by the prediction (Fig 1), then the corresponding helix would have to be of at least 30 residues long This implies in turn that the M10 segment of PMA1_NEUCR would end before residue 890 It is worth noting that the M9 and M10 helices of this enzyme were detected by all algorithms with a high level of confidence and were accordingly superimposed to their structural equivalents in ATC1_RABIT The final sequence alignment is shown in Fig with a comparison between the secondary structure predictions for PMA1_NEUCR sequence and the assigned secondary structure of the 3D model Although this secondary structure prediction was not used in the alignment part of the modeling procedure, it is presented here to show that in the cytosolic domains, the secondary structure prediction and the secondary structure resulting from the modeling procedure are remarkably consistent Position of PMA1_NEUCR and ATC1_RABIT transmembrane segments are indicated as shaded colored boxes Model building Using the sequence alignment displayed in ˚ Fig and the 2.6 A resolution X-ray structure of ATC1_RABIT (RSCB-pdb code: 1EUL) [8], a first model of PMA1_NEUCR was generated with the PromodII [29] package of DEEPVIEW 3.7 [30] Reconstruction of the loops in gap regions was achieved with the loop database module of DEEPVIEW Model quality assessment and refinement The quality of the model was assessed using the WHATIF ˚ v.4.99 [31] and PROCHECK [32] validation suites The 2.6 A resolution structure of ATC1_RABIT was analyzed with the same suites as a control The results showed that the bonded geometry and the stereochemistry of both structures 5250 O Radresa et al (Eur J Biochem 269) were of similar quality, indicating that care was taken in both the homology modeling and X-ray refinement procedures to optimize these parameters However, significant differences were observed in the number of close nonbonded contacts Those were higher in the constructed model of PMA1_NEUCR than for the crystal structure of the rabbit enzyme To relieve this strain the model was subjected to two short energy minimization runs with GROMOS 96 [33] using the GROMOS 43B1 force field These runs involved 200 steps of steepest descent followed by 300 steps of conjugate gradient optimizations This led to a significant drop in the unfavorable nonbonded contacts, while producing only very minor displacements of the atomic coordinates Identification of internal cavities Internal cavities were identified from the atomic coordinates of the PMA1_NEUCR model, ATC1_RABIT (1EUL.pdb), bacteriorhodopsin (1C3W.pdb), halorhodopsin (1E12.pdb), L-2-haloacid dehalogenase (1JUD.pdb) and phosphoserine phosphatase (1F5S.pdb) using the surface module of DEEPVIEW 3.7 and the program SURVOL [34] In both programs the computed cavities were delimited by ˚ the molecular surface computed with a probe size of 1.4 A RESULTS AND DISCUSSION Comparison of the ion binding sites in ATC1_RABIT with the equivalent region in PMA1_NEUCR In ATC1_RABIT and other mammalians P-type ATPases [35,36], several amino acids involved in cation binding were identified by site-directed mutagenesis along transmembrane segments M4, M5, M6 and M8 The crystal structure of ATC1_RABIT reveals how these residues assemble to form a binding pocket surrounding two Ca2+ ions A comparison of this binding pocket with the corresponding domain of our 3D model of PMA1_NEUCR is shown in Fig Ion binding site I Five residues form the first Ca2+ binding site The calcium ion is bound to the side-chain oxygen of Asn768 and Glu771 on M5, of Thr799 and Asp800 on M6 and Glu908 on M8 All the oxygens are arranged in roughly the same plane except for the side chain of Glu771, which is located below Ó FEBS 2002 In our PMA1_NEUCR model, these residues are replaced by Ser699, Leu702 on M5, by Ala729 and the conserved Asp730 on M6, and Ala814 on M8 With this residue constellation, the ion-binding site is most probably lost The possibility remains, however, that the presence of Ser and Asp residues in this region would allow it to participate in proton transport Alanine-scanning mutagenesis along M5 [37] showed that Ser699 is probably involved in proton transport, although it is not essential to the coupling mechanism Substitution of Leu702 to Ala resulted in an enzyme with a normal coupling ratio Replacement of Asp730 a conserved residue in segment M6 by Asn or Val led to a poorly folded enzyme, arrested in the endoplasmic reticulum [41] Nevertheless, double mutants in which both charged Arg695 and Asp730 were inversed or replaced by Ala reverted to a fully functional enzyme These observations indicate that these residues are linked by a salt bridge, making a direct participation in proton transport unlikely [39] In plant H+-ATPases (Arabidhopsis thaliana), however, the conserved Asp residue does not seem to be involved in a salt bridge and might hence play a role in proton transport [40] Nevertheless, among the investigated residues of yeast and fungi H+-ATPases corresponding to calcium binding site of ATC1_RABIT, only Ser699 seems to play a role, albeit a nonessential one, in proton transfer Ion binding site II The second calcium-binding site of ATC1_RABIT is formed by six residues, nearly all of which are located on M4 This site is formed by main-chain carbonyl oxygen of Val304, Ala305, and Ile307 on M4; by side-chain oxygens of Glu309 on M4 and of Asn796 and Asp800 on M6 In our PMA1_NEUCR model, these residues correspond to Ile331, Ile332, Val334, and Val336 on M4 and Ala726 and Asp730 on M6 Alanine-scanning mutagenesis along segment M4 of yeast H+-ATPase showed that replacement of Ile331 and Val334 had little or no effect on ATP-dependent proton transport [41], not inconsistent with the fact that the mutations not change the nature of the backbone Replacement of Ile332 or Val336 resulted, however, in a coupled mutant enzyme displaying altered kinetics consistent with a slow down of the E1P–E2P transition step believed to be coupled to the charge transfer reaction Finally, Asp730 corresponding to Asp800 in ATC1_RABIT was shown to be involved in a salt bridge with Arg695 precluding direct participation in proton transport, as discussed above Fig Comparison of ATC1_RABIT (A) and PMA1_NEUCR (B) ion-binding sites regions Calcium ions in ATC1_RABIT are labeled according to the binding site numbers A possible conformation for Arg695 and Asp730 making a salt bridge between M5 and M6 is indicated by the dotted line Ó FEBS 2002 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5251 In conclusion, comparisons of the residues involved in ATC1_RABIT calcium binding sites with their structural equivalents in PMA1_NEUCR indicate clearly that this region is not conserved in the latter enzyme However, as it appears from the mutagenesis studies described above, the possibility that some residues in this region are involved in proton transfer can not be ruled out Only three residues (Ser699, Ile332 and Val336) out of the 10 residues equivalent to the cation binding residues in ATC1_RABIT might play a role in this process, but probably not an essential one Identification of a putative proton pathway in Neurospora crassa H+-ATPase The chemiosmotic model for PMA1_NEUCR In the P-type proton pumps, the origin of the transported proton as well as the proton entry pathway is still unknown The socalled Ôchemiosmotic modelÕ for PMA1_NEUCR, based largely on biochemical data, makes an interesting proposal concerning the initiation site for proton transport [49] It stipulates that this transport is initiated by the lysis of a water molecule in the cytoplasmic phosphorylation site, implying that the proton pathway would begin close to the phosphorylated aspartate (Asp378) The major steps of the model are as follows: The first step is a covalent phosphoryl-transfer reaction from the MgATP molecule to the strictly conserved Asp residue (Asp378), as shown by radioactively labeled ATP [42,43] The next step is dephosphorylation of the aspartylphosphate group with subsequent release of phosphate at the cytoplasmic side of the enzyme This reaction involves a water molecule whose oxygen atom promotes disruption of the covalent bond between the conserved Asp378 residue and Pi [44] The released protons have been proposed to be withdrawn by functional residues acting as general bases on their way to the transport reaction [49] Internal polar cavities as loci of proton transport in membrane proteins In monomeric and globular proteins, internal polar cavities often contain buried water molecules which interact both with other protein groups and with one another [11–13] Such cavities could therefore represent loci where water molecules could form H-bond networks that foster proton conduction [46,48] On the basis of this hypothesis, we set out to identify a possible pathway for proton transport in PMA1_NEUCR by identifying the position of internal polar cavities in the built molecular model The role of water molecules as a crucial determinant of the proton conduction network has been amply documented in bacteriorhodopsin [45] To lend support to our hypothesis, we therefore applied the same approach to the high-resolution structure of bacteriorhodopsin and the closely related structure of halorhodopsin, which are the two highest resolution structures for any transmembrane protein to date Position of internal polar cavities in the high-resolution structures of halo- and bacteriorhodopsin Using the procedure described in the Methods section, we identified ˚ the internal polar cavities in the 1.55 A resolution structure ˚ of bacteriorhodopsin (1C3W.pdb) and the 1.8 A resolution structure of halorhodopsin (1E12.pdb) In both structures, essentially all the buried crystallographic water molecules are located in internal polar cavities, as illustrated in Fig 3A,B [46,47] In the case of the bacteriorhodopsin proton pump, eight such cavities have been identified The 15 polar residues that line these cavities are Thr5, Arg7, Thr46, Tyr57, Tyr79, Arg82, Tyr83, Asp96, Ser193, Glu194, Glu204, Tyr205, Asp212, and Lys216 Of these, 10 residues (Thr46, Tyr57, Arg82, Asp85, Asp96, Glu194, Glu204, Glu205, Asp212 and Lys216) are believed to be involved in the network of the hydrogen-bonded residues and water molecules that define the proton pathway [48] Thus, in this case, determining the position of internal polar cavities in the 3D structure enable to outline the pathway for proton transfer Identifying such cavities in the model of PMA1_NEUCR, a membrane protein for which the proton pathway has not as yet been delineated might provide useful information about this pathway Positions of internal polar cavities in the PMA1_NEUCR model Applying the procedure of cavity identification to the PMA1_NEUCR model yields a total of 21 internal ˚ cavities, whose volume varies from 14 to 93 A3 Most of them are located along the longitudinal axis of the protein as seen in Fig Along this axis, two main groups of cavities can be distinguished The first is located just below the cytoplasmic site of MgATP hydrolysis The second group, located in the membrane domain, almost connects the region homologous to the ion-binding site to the extracytoplasmic moiety of the molecule The positions of the internal polar cavities hence suggest that the proton translocation pathway might begin close to the phosphorylation site in the large cytoplasmic loop, in agreement with the semi-empirical chemiosmotic model for PMA1_NEUCR [49] In order to verify this suggestion, we listed all the residues lining these cavities and compared them to those shown to affect the proton transport reaction by mutagenesis experiments The cavity-lining residues number 54 in all, of which 22 are identical to those in ATC1_RABIT (see alignment of Fig 1) Table lists the 54 residues, together with the cavity they contact, and the effect of mutations on the kinetic properties and the coupling ratio between ATPase activity and proton transport These residues are numbered on the sequence alignment of Fig 1, in italic fonts when a mutation has been reported or in regular font when they have not yet been investigated (a) Internal polar cavities in the transmembrane domain In the transmembrane domain, the first small cavity (cavity 7) is located between Val289 and Ile293 in the N-terminal end of M3, and Trp756 and Gly757 in the cytoplasmic loop connecting M6 and M7 As shown by mutagenesis in yeast, mutation of Val289 to Phe resulted in an altered phenotype that seems not to be directly related to the charge translocation step [50] In ATC1_RABIT, however, the homologous residues were identified as possible determinants for control of the gateway to the Ca2+ binding sites by site-directed mutagenesis [51,52] The next cavity in the transmembrane domain is located above the ion-binding site region (cavity 8) Important residues in contact with this cavity are Tyr691 and Val336 Replacement of Tyr691 by Ala resulted in an enzyme 5252 O Radresa et al (Eur J Biochem 269) Ó FEBS 2002 Fig Position of internal polar cavities and crystallographic water molecules in the high resolution crystal structures of bacteriorhodopsin (A) and halorhodopsin (B) Red spheres represent buried crystallographic water molecules; blue dots represent crystallographic water molecules lying outside the surface envelope of the enzymes Cavities are colored by atom type (red stands for O, blue for N and yellow for S) The backbone position of the ten polar residues lining the proton pathway and contacting the identified internal polar cavities is colored in green Fig Position of internal polar cavities in PMA1_NEUCR model (A) and (B) views and numbering of internal polar cavities The view in (B) is rotated by nearly 90° with respect to (A) (the NH2-terminus is omitted for clarity) Cavities are colored by atom type (red stands for O, blue for N and yellow for S) The horizontal black lines delineate the approximate membrane position The catalytic Asp (Asp378) appears in red defective in the E1-E2 conformational change Here, the Val336Ala mutant displayed kinetic properties consistent with a decrease of the transport-linked E1P–E2P transition step [41] As seen above, Val336 corresponds to Glu309 of ATC1_RABIT that contributes directly to calcium binding site (Fig 2) The following long cavity (cavity 9) is located between helices M4, M5, M6 and M8 again in the region corresponding to the ion-binding site A conserved residue, Tyr694, is making contact with this cavity The Tyr694 to Ala mutant strongly decreased ATPase activity, while the Tyr694 to Gly mutant displayed a strong resistance towards inhibition by vanadate Interestingly, Tyr694Ala mutant resulted in a presumably uncoupled enzyme, although the low rate of ATP hydrolysis prevented a detailed analysis of the coupling ratio In ATC1_RABIT, mutation of the corresponding residue (Tyr763), clearly led to an uncoupled enzyme unable to transport Ca2+ ions [53] Another residue in contact with cavity is Ser699 that was found to be involved in proton translocation (see above) Two other cavities are observed between M6, M8 and M9 that are in the nonhomologous C-terminal domain built using the topology-based alignment (see Fig 1) The position of the side chains and hence of cavities in this domain are therefore considered as less reliable than in other parts of the model It is nonetheless of interest that residue Glu805 corresponding to Glu803 in the yeast H+-ATPase is found close to cavity number 10, a small internal cavity that appears as nonpolar in our model Nevertheless mutagenic studies in yeast H+-ATPase revealed that substitution at Glu803 by either Gln or Asn seems to increase the rate proton transport [38] In our model, this residue is partially accessible to the solvent and may therefore act as a gate for the possible exit of a watercoordinated proton occurring at the extra-cytoplasmic end of the proton transfer path 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5253 Ó FEBS 2002 Table List of the residues in contact with the cavities to The effect of a mutation is reported when the data was available (a) Observed but not characterized due to low rate of ATP hydrolysis ND, not determined Region Cavity Rossman fold Cavity Rossman fold Mutation Expression Coupling Kinetic E1–E2 ATPase activity K615 M631 T632 G633 D638 S641 L642 – – – – – – – – Lethality – – – – – – – – – – – – – – ND ND – – – – – – ND ND Misfolded Misfolded – – – – 94% 15% – – – – – – [61] [54] Misfolded – – – – – – – – – – ND – – – – – – – – – – [54] – – – – – – – – – – Misfolded ND ND – – – – – – ND ND – – – – – – 29% 94% 15% – – – – – – [54] [61] [54] 86% (a) – – – – – – – – Altered – – – – – – – – 12% – – – – – – – – [55] 96% 37% 85% 44% 84% 54% 86% Normal ND Normal ND Normal Normal ND – – – Altered Normal Normal Normal Altered Altered Altered – – – 55% 23% 123% 36% 45% 77% 12% – – – [41] [41] [41] [41] [41] [55] [55] C376 S377 D378 N – – A A N, S E L557 K615 M631 T632 Cavity Rossman fold Cavity Rossman fold Cavity Rossman fold Stalk M5 Cavity Stalk M4 Stalk M5 T382 T632 G633 I649 A650 V651 F666 P669 G670 L671 I674 A A A L369 A630 T647 I649 I664 F666 I674 A677 L678 A M346 G349 A350 L353 V360 I366 L369 A677 T680 S681 – – – – – – A L375 C376 S377 A630 M631 T632 I649 L671 I674 104% 103% 14% A A S A A A A – – – – – – – – – – 31% 104% 103% – – – – – – – – – – – – – – – – – Reference [60] [62] Ó FEBS 2002 5254 O Radresa et al (Eur J Biochem 269) Table (Continued) Region Mutation Cavity TM3 V289 L67 Cavity TM4 TM5 TM6 Expression I293 W756 G757 P335 V336 Y691 R695 A A A A D730 D N, V E L734 Cavity TM4 TM5 TM5 TM6 TM8 I332 Y694 R695 L698 S699 H701 L702 I725 A726 A729 D730 L734 F810 A A A A A C T A A N, V E Kinetic E1–E2 ATPase activity Reference ND ND [50] – – – Altered revertant of S368F – – – 103% – – – F Coupling 80% 108% 92% 14% < 10% < 10% – 71% 40% 14% 90% 90% 79% 94% 15% 39% – – – < 10% < 10% – – (a) Normal ND ND – Normal (a) Normal (a) Normal Normal ND ND – – – Misfolded ND – – We thus see that the pathway outlined by the cavities numbered 7–10 connects the bottom of the phosphorylation site organized in a Rossman fold to the extracytoplasmic moiety Furthermore, all these cavities, with the exception of cavity 7, are lined by one or more residues previously shown to be involved in the proton translocation step (b) Internal polar cavities in the cytoplasmic domain The cavities identified in this domain trace a clear path that starts in the center of the b-strands forming the Rossman fold and leads to the membrane domain at the top of M4 and M5 (see Fig 4, cavities numbered 1–6) As could be expected from the high level of sequence identity around the phosphorylated aspartate (Asp378 in PMA1_NEUCR), several cavities are also seen at the same position in ATC1_RABIT (data not shown) Aside from mutations directed against a small stretch of amino acids adjacent to the phosphorylated Asp [54], limited information is available from mutagenesis studies on residues involved in proton translocation in the cytoplasmic phosphorylation site At the bottom of the cytoplasmic domain, cavity number is in contact with two residues of interest for the proton translocation step: Met346 and I366 Mutations of either Met346 or Ile366 to Ala result in a reduced Km for MgATP, Normal Altered Altered Misfolded salt bridge with D730 revertant of D730R Misfolded ND – Altered Altered Misfolded Normal Normal pH shift Normal ND Altered – – – Unactive ND – – – – – 22% 96% 0% Unactive 15% – 32% 22% 70% 8% 16% 96% Unactive 42% – – – 15% – – [41] [41] [37] [37] [39] [39] [39] [38] [41] [37] [37] [37] [37] [37] [37] [37] [37] [39] [38] an acidic shift of the optimum pH and an increased resistance towards inhibition by vanadate These effects are consistent with a slow down of the transport-linked E1P– E2P transition step, suggesting that they may be involved in the transport reaction [41,55] An apparent pathway by which a proton might be internalized from the cytosol to reach cavity is through cavities 1–5 Indeed, polar residues are lining these cavities, consistent with the idea that they might harbor a watermediated H-bond network fostering proton transfer However, a potential problem in validating this proposal is that the fold of PMA1_NEUCR seems very sensitive to mutations directed against residues adjacent to the catalytic Asp378 Nevertheless, most of the residues lining cavities 1–5 are found in the vicinity of the ÔAMTGDGVNDAP640Õ motif, a region that has as yet not been thoroughly investigated The residues identified in this region (Table 3) might thus constitute suitable targets for site-directed mutagenesis (c) Internal polar cavities in soluble members of the haloacid dehalogenase superfamily (HAD) With little information on the residues lining our proposed entry pathway available from mutagenesis studies, positions of the cavities located in our model were compared with those Ó FEBS 2002 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5255 of internal cavities capable of containing water molecules in the homologous Rossman fold of the soluble members of the HAD superfamily (Fig 5) We used the high-resolution structures of the L-2-haloacid dehalogenase (1JUD) and of phosphoserine phosphatase (1J5S) that exhibit the same organization of the active site (Rossman fold) in the cytoplasmic domain of the P-type ATPases In phosphoserine phosphatase and L-2-haloacid dehalogenase structures, respectively, 69% and 66% of the Ca comprising the Rossman fold displayed an root mean ˚ squared deviation of less than 1.85 A with the homologous domain of PMA1_NEUCR model This makes them close structural homologues of the cytoplasmic phosphorylation site in the P-type ATPases as has already been reported elsewhere [56–59] Internal polar cavities and crystallographic water molecules were identified in these structures using the described procedure Quite strikingly, in both cases, the structures exhibit a single internal polar cavity in the vicinity of the conserved catalytic Asp This cavity contains two to three crystallographic water molecules In stark contrast, the Rossman fold of PMA1_NEUCR contains as many as six polar cavities, which link the phosphorylated Asp378 all through the middle of the a/b Rossman fold, down to the Fig Position of internal polar cavities in the Rossman fold of PMA1_NEUCR and two other soluble members of the HAD superfamily (A) Phosphorylation site of PMA1_NEUCR, (B) phosphorylation site of Phosphoserine phosphatase, (C) active site of L-2-haloacid dehalogenase The catalytic Asp in the Rossman fold appears in red Red spheres represent buried crystallographic water molecules; blue dots represent crystallographic water molecules lying outside the surface envelope of the enzymes Cavities are colored by atom type (red stands for O, blue for N and yellow for S) beginning of the transmembrane domain The presence of this string of polar cavities in PMA1_NEUCR Rossman fold, the most accurate part of our model, and its conspicuous absence in the otherwise close structural relatives from the soluble members of the HAD family, point to a possible functional role of these cavities in PMA1_NEUCR CONCLUSION In this study, we built a complete atomic model of Neurospora crassa plasma membrane H+-ATPase (PMA1_NEUCR) using the high-resolution 3D structure of the rabbit sarcoplasmic reticulum Ca2+-ATPase (ATC1_RABIT) as a template and combining transmembrane topology predictions with classical homology modeling techniques in regions of low sequence similarity In the first part of this work, the comparison of the ionbinding site of ATC1_RABIT with the homologous domain in PMA1_NEUCR revealed that this domain is not conserved in both enzymes This suggests that although the P-type ATPases are widely assumed to share a common mechanism of action, each group of proteins probably Ó FEBS 2002 5256 O Radresa et al (Eur J Biochem 269) possesses some specific chemical and structural features enabling them to fulfill their specialized transport function In the second part of this work, the model of PMA1_NEUCR was used to predict a proton transport pathway that would start near the phosphorylation site at the cytoplasmic domain, pass through the transmembrane region and end at the extracytoplasmic side This pathway was predicted by identifying internal cavities lined by polar residues, an original approach that we validate by showing that it is capable of identifying the residues involved in proton transport in the high-resolution structure of bacteriorhodopsin, without any prior information Some of the residues lining the identified cavities in PMA1_NEUCR were shown previously to be involved in proton transfer by mutagenesis experiments, whereas the role of others has as yet not been assessed This concerns in particular polar residues such as Thr382, Thr632, Thr647, Thr680, Ser377, Ser641, Ser681, or Lys615 located along the proposed proton entry pathway starting near the phosphorylation site, which is the most reliable portion of our 3D model Given the overall low sequence similarity between the H+- and Ca2+-ATPasse, the 3D structure built here represents, for the most part, a low resolution model for the H+-ATPase, in which side-chain positions and hence internal cavities are probably determined with limited accuracy This is particularly true for the cavities located in the last segments of the transmembrane domain, hence these cavities have been discussed in light of several independent mutational studies On the other hand, the phosphorylation domain where we locate the proposed proton entry pathway is a much more reliable portion of our model, as the sequences of the eukaryotic P-type ATPases are essentially conserved in this domain Moreover, we illustrated that the fold of this domain is remarkably conserved between the Ca2+-ATPase and the soluble members of the haloacid dehalogenases whose catalytic activity proceed essentially by the same mechanism Indeed, catalytic residues occupy the same position in the 3D structures of the two enzymes In addition, the predicted secondary structures for several P-type ATPases including the H+-ATPase are also remarkably well conserved in this domain Likewise, the secondary structure of the phorphorylation domain deduced from the model built here using the Ca2+-ATPase as template and that determined directly from the amino acid sequence of the H+-ATPase, are also in excellent agreement All this indicates that the tertiary structure of this Rossman fold domain is very probably quite well conserved among the P-type ATPase proteins We therefore believe that this domain is the most accurate part of our model, warranting some confidence in the proposed proton entry pathway, which is mostly located in this domain A good indication that the delineated pathway might be relevant to function is our finding that a similar pathway cannot be traced in the structurally related soluble members of the HAD super-family, which unlike PMA1_NEUCR not mediate proton translocation The proton transport pathway proposed here is consistent with the semiempirical chemiosmotic model for proton transport in PMA1_NEUCR However, while it is not inconsistent with some of the suggestions of Bukrinsky et al [11], namely that the region of PMA1_NEUCR equivalent to the second cation binding site of ATC1_RABIT might stabilize a hydronium ion, we not believe that such ion is the transported species We favor instead the well documented mechanism of proton conduction through a network of hydrogen-bonded internal water molecules interacting with key positioned polar groups, described in other systems [45,46,48] Involvement of the identified polar residues lining the proposed proton entry path requires now proper experimental validation Application of mutagenesis and, better, a combination of mutagenesis and high-resolution structural analyses on PMA1_NEUCR, or a close homolog with the same function, will certainly provide important clues on the molecular mechanism used by these complex proton pumps ACKNOWLEDGMENTS ` O R is recipient of the ÔFonds pour la Formation a la Recherche dans lÕIndustrie et dans l’Agriculture’ (Belgium) E G is Research Director of the ÔFonds National de la Recherche ScientifiqueÕ (Belgium) We ´ thank the ÔCommunaute Francaise de Belgique: Actions de Recherche ¸ ´ ConcerteesÕ for financial support REFERENCES Axelsen, K.B & Palmgren, M.G (1998) Evolution of substrate specificities in the P-type ATPase superfamily J Mol Evol 46, 84–101 Slayman, C.L., Long, W.S & Lu, C.Y (1973) The relationship between ATP and an electrogenic pump in the plasma membrane of Neurospora crassa J Membr Biol 14, 305–338 Goormaghtigh, E., Chadwick, C & Scarborough, G.A (1986) Monomers of the Neurospora plasma membrane catalyze efficient proton translocation J Biol Chem 261, 7466–7471 Auer, M., Scarborough, G.A & Kuhlbrandt, W (1998) Threedimensional map of the plasma membrane H+-ATPase in the open conformation Nature 392, 840–843 Zhang, P., Toyoshima, C., Yonekura, K., Green, N.M & Stokes, D.L (1998) Structure of the calcium pump from sarcoplasmic ˚ reticulum at 8A resolution Nature 392, 835–839 Scarborough, G.A (1999) Structure and function of the P-type ATPases Curr Opin Cell Biol 11, 517–522 Stokes, D.L., Auer, M., Zhang, P & Kuhlbrandt, W (1999) Comparison of H+-ATPase and Ca2+-ATPase suggests that a large conformational change initiates P-type ion pump reaction cycles Curr Biol 9, 672–679 Toyoshima, C., Nakasako, M., Nomura, H & Ogawa, H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum ˚ at 2.6 A resolution Nature 405, 647–655 Bukrinsky, J.T., Buch-Pedersen, M.J., Larsen, S & Palmgren, M.G (2001) A putative proton-binding site of plasma membrane H+-ATPase identified through homology modelling FEBS Lett 494, 6–10 10 Rammelsberg, R., Huhn, G., Lubben, M & Gerwert, K (1998) Bacteriorhodopsin’s intramolecular proton-release pathway consists of a hydrogen-bonded network Biochemistry 37, 5001– 5009 11 Williams, M.A., Goodfellow, J.M & Thornton, J.M (1994) Buried waters and internal cavities in monomeric proteins Protein Sci 4, 1224–1235 12 Hubbard, S.J., Gross, K.H & Argos, P (1994) Intramolecular cavities in globular proteins Protein Eng 7, 613–626 13 Rashin, A.A., Iofin, M & Honig, B (1986) Internal cavities and buried waters in globular proteins Biochemistry 25, 3619– 3625 14 Cserzo, M., Wallin, E., Simon, I., von Heijne, G & Elofsson, A (1997) Prediction of transmembrane alpha-helices in procariotic Ó FEBS 2002 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 3D modelling of Neurospora H+-ATPase (Eur J Biochem 269) 5257 membrane proteins: the dense alignment surface method Protein Eng 10, 673–676 Rost, B., Fariselli, P & Casadio, R (1996) Topology prediction for helical transmembrane proteins at 86% accuracy Protein Sci 7, 1704–1718 ´ Tusnady, G.E & Simon, I (1998) Principles governing amino acid composition of integral membrane proteins: applications to topology prediction J Mol Biol 283, 489–506 Sonnhammer, E.L.L., von Heijne, G & Krogh, A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences In Proceedings of the of Sixth International Conference on Intelligent Systems for Molecular Biology (Glasgow J., Littlejohn T., Major F., Lathrop R., Sankoff D and Sensen C., Eds), pp 175–182 AAAI Press, Menlo Park, CA Hofmann, K & Stoffel, W (1993) TMbase – a database of membrane spanning proteins segments Biol Chem Hoppe-Seyler 347, 166–171 Lin, J & Addison, R (1994) Topology of the Neurospora plasma membrane H+-ATPase Localization of a transmembrane segment J Biol Chem 269, 3887–3890 Lin, J & Addison, R (1995) The membrane topology of the carboxyl-terminal third of the Neurospora plasma membrane H+ATPase J Biol Chem 270, 6942–6948 Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M & Barton, G.J (1998) Jpred: a consensus secondary structure prediction server Bioinformatics 14, 892–893 Appel, R.D., Bairoch, A & Hochstrasser, D.F (1994) A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server Trends Biochem Sci 19, 258–260 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680 Huang, X & Miller, W (1991) A time-efficient, linear-space local similarity algorithm Adv Appl Math 12, 337–357 Rost, B & Sander, C (1994) Combining evolutionary information and neural networks to predict protein secondary structure Proteins 19, 55–72 Moller, J.P., Juul, B & le Maire, M (1996) Structural organization, ion transport and energy transduction of P-type ATPases Biochim Biophys Acta 1286, 1–51 Rao, U.S., Hennessey, J.P Jr & Scarborough, G.A (1991) Identification of the membrane embedded regions of Neurospora crassa plasma membrane H+-ATPase J Biol Chem 266, 14740–14746 Portillo, F (2000) Regulation of plasma membrane H+-ATPase in fungi and plants Biochim Biophys Acta 1469, 31–42 Peitsch, M.C & Guex, N (1996) Promod and Swiss-model internet based tools for automated comparative protein modelling Biochem Soc Trans 24, 274–279 Guex, N & Peitsch, M.C (1997) SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modelling Electrophoresis 18, 2714–2723 Hooft, R.W.W., Vriend, G., Sander, C & Abola, E.E (1996) Errors in protein structures Nature 381, 272–274 Laskowski, R.A., MacArthur, M.W., Moss, D.S & Thornton, J.M (1993) PROCHECK: a program to check the stereochemical quality of protein structures J Appl Cryst 26, 283–291 van Gunsteren, W.F., Billeter, S.R., Eising, A.A., Hunenberger, ă P.H., Kruger, P., Mark, A.E., Scott, W.R.P & Tironi, I.G ă (1996) Biomolecular Simulation: the GROMOS96 Manual and User Guide Vdf Hochschulverlag AG an der ETH Zu ărich, pp 11042 Zurich, Switzerland Pontius, J., Richelle, J & Wodak, S.J (1996) Quality assessment of protein 3D structures using standard atomic volumes J Mol Biol 264, 121–136 35 Clarke, D.M., Loo, T.W., Inesi, G & MacLennan, D.H (1989) Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+ATPase Nature 339, 476–478 36 Jorgensen, P.L & Pedersen, P.A (2001) Structure–function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase Biophys Biochim Acta 1505, 57–74 37 Dutra, M.B., Ambesi, A & Slayman, C.W (1998) Structurefunction relationship in membrane segment five of the yeast PMA1 H+-ATPase J Biol Chem 273, 17411–17417 38 Petrov, V.V., Padmanhaba, K.P., Nakamoto, R.K., Allen, K.E & Slayman, C.W (2000) Functional role of charged residues in the transmembrane segments of the yeast H+-ATPase J Biol Chem 275, 15709–15716 39 Gupta, S.S., DeWitt, N., Allen, K.E & Slayman, C.W (1998) Evidence for a salt bridge between transmembrane segments and of the yeast plasma-membrane H+-ATPase J Biol Chem 273, 34328–34334 40 Buch-Pedersen, M.J., Venema, K., Serrano, R & Palmgren, M.G (2000) Abolishment of proton pumping and accumulation in the E1-P conformational state of a plant plasma membrane H+ATPase by substitution of a conserved aspartyl residue in transmembrane segment J Biol Chem 275, 39167–39173 41 Ambesi, A., Pan, R.L & Slayman, C.W (1996) Alanine scanning mutagenesis along membrane segment four of the yeast plasma membrane H+-ATPase Effect on structure and function J Biol Chem 271, 22999–23005 42 Dame, J.B & Scarborough, G.A (1981) Idenfication of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as a beta-aspartyl phosphate J Biol Chem 256, 10724–10730 43 Dame, J.B & Scarborough, G.A (1980) Identification of the hydrolytic moiety of the Neurospora plasma membrane H+-ATPase and demonstration of a phosphoryl-enzyme intermediate in its catalytic mechanism Biochemistry 19, 2931–2937 44 Amory, A., Goffeau, A., McIntosh, D.B & Boyer, P.D (1982) Exchange of oxygen between phosphate and water catalyzed by the plasma membrane ATPase from the yeast Schizosaccharomyces pombe J Biol Chem 257, 12509–12516 45 Kandori, H (2000) Role of internal water molecules in bacteriorhodopsin Biochim Biophys Acta 1460, 177–191 46 Luecke, H., Schobert, B., Richter, H.T., Cartailler, J.P & Lanyi, ˚ J.K (1999) Structure of bacteriorhodopsin at 1.55 A resolution J Mol Biol 291, 899–911 47 Kolbe, M., Besir, H., Essen, L.-O & Oesterhelt, D (2000) ˚ Structure of light-driven chloride pump halorhodopsin at 1.8 A resolution Science 288, 1390–1396 48 Luecke, H., Richter, H.-T & Lanyi, J.K (1998) Proton transfer ˚ pathways in bacteriorhodopsin at 2.3 A resolution Science 280, 1934–1937 49 Scarborough, G.A (1982) Chemiosmotic models for the mechanisms of the cation-motive ATPases Ann NY Acad Sci 402, 99– 115 50 Harris, S.L., Perlin, D.S., Seto-Young, D & Haber, J.E (1991) Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H+-ATPase J Biol Chem 266, 24439–24445 51 Andersen, J.P., Sorensen, T.L.-M., Povlsen, K & Vilsen, B (2001) Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites J Biol Chem 276, 23312–23321 52 Menguy, T., Corre, F., Bouneau, L., Deschamps, S., Moller, J.V., Champeil, P., le Maire, M & Falson, P (1998) The cytoplasmic loop located between transmembrane segments and controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase J Biol Chem 273, 20134–20143 5258 O Radresa et al (Eur J Biochem 269) 53 Andersen, J.P (1995) Functional consequences of alterations to amino acids at the M5S5 boundary of Ca2+-ATPase of sarcoplasmic reticulum J Biol Chem 270, 908–914 54 DeWitt, N.D., Tourinho dos Santos, C.F., Allen, K.E & Slayman, C.W (1998) Phosphorylation region of the yeast plasmamembrane H+-ATPase Role in protein folding and biogenesis J Biol Chem 273, 21744–21751 55 Ambesi, A., Miranda, M., Allen, K.E & Slayman, C.W (2000) Stalk segment of the yeast plasma membrane H+-ATPase Mutational evidence for a role in the E1–E2 conformational change J Biol Chem 275, 20545–22050 56 Wang, W., Kim, R., Jancarik, J., Yokota, H & Kim, S.-H (2001) Crystal structure of phosphoserine phosphatase from Methano˚ coccus jannaschii, a hyperthermophile, at 1.8 A resolution Struct Fold Design 9, 65–72 57 Koonin, E.V & Tatusov, R.L (1994) Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity Application of an iterative approach to database search J Mol Biol 244, 125–132 Ó FEBS 2002 58 Aravind, L., Galperin, M.Y & Koonin, E.V (1998) The catalytic domain of the P-type ATPase has the haloacid dehalogenase fold Trends Biol Sci 23, 127–129 59 Stokes, D.L & Green, N.M (2000) Modeling a dehalogenase fold ˚ into the 8-A density map for Ca2+-ATPase defines a new domain structure Biophys J 78, 1765–1776 60 Portillo, F (1997) Characterization of dominant lethal mutations in the yeast plasma membrane H+-ATPase gene FEBS Lett 402, 136–140 61 Petrov, V.V & Slayman, C.W (1995) Site-directed mutagenesis of the yeast PMA1 H+-ATPase Structural and functional role of cysteine residues J Biol Chem 270, 28535–28540 62 Nakamoto, R.K., Verjovski-Almeida, S., Allen, K.E., Ambesi, A., Rao, R & Slayman, C.W (1998) Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis J Biol Chem 273, 7338–7344 63 Wolfgang Kabsch, W & Sander, C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features Biopolymers 22, 2577–2637  ... probably not an essential one Identification of a putative proton pathway in Neurospora crassa H+-ATPase The chemiosmotic model for PMA1_NEUCR In the P-type proton pumps, the origin of the transported... ATP hydrolysis were built for plant and yeast H+-ATPases on the basis of the ACT1_RABIT crystal structure [9] From these models is was proposed that proton transport in the H+-ATPases is mediated... extracytoplasmic side This pathway was predicted by identifying internal cavities lined by polar residues, an original approach that we validate by showing that it is capable of identifying the