Modeling the three-dimensional structure of H
+
-ATPase
of
Neurospora crassa
Proposal for a proton pathway from the analysis of internal cavities
Olivier Radresa
1
, Koji Ogata
2
, Shoshana Wodak
2
, Jean-Marie Ruysschaert
1
and Erik Goormaghtigh
1
1
Service de Structure et Fonction des Membranes Biologiques, Universite
´
Libre de Bruxelles, Bruxelles, Belgium;
2
Unite
´
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

-ATPase model, most of these cavities are in contact with
residues previously shown t o a ffect coupling o f p roton
translocation to ATP hydrolysis. A string of six polar cavi-
ties 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 halo-
acid dehalogenase superfamily, which are close structural
homologs of this domain but do not shar e t he same function,
display only one polar cavity in the vicinity of the conserved
catalytic Asp re sidue.
Keywords: neurospora; P-ATPase; homology model; cavity;
H
+
.
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 re mains
indeed a m ajor limiting step for X-ray or electron crystal-
lography. Alternative approaches for obtaining structural
information are therefore very useful. One such approach is
the homology modeling t echnique whereb y a known 3D
structure of a related protein is used as a template for
building a n a tomic model from the amino acid sequence of
the target protein. Although validity of the resulting m odel
requires experimental confirmation , it c an provide u seful
insights into the structure–function relationship in related
enzymes.
TheplasmamembraneH
+
-ATPase of t he fungi Neuro-

Recently, the resolution of the structure of ATC1_
RABIT w as increased to 2 .6 A
˚
providing t he 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: [email protected]
Abbreviations: HAD, haloacid dehalogenase; TM, M , trans-
membrane segment; PSP, phosphoserine phosphatase.
Enzymes: PMA1_NEUCR, Neurospora crassa plasma-membrane
H
+
-ATPase; ATC1_RABIT, Oryctolagus cuniculus (rabbit) Ca
2+
-
ATPase of sarcoplasmic reticulum (splice isoform SERCA1a).
(Received 27 May 2002, revised 23 A ugust 2002,
accepted 6 S eptember 2002)
Eur. J. Biochem. 269, 5246–5258 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03236.x
structure obtained in presence of two buried calcium ions is
believed to represent an open conformational state analog-
ous to the previously determined 8 A
˚
resolution
PMA1_NEUCR structure.
This, together with the striking similarities between the

the p rotein and a networ k o f interacting buried or partially
buried water molecules [10]. To find pathways consistent
with this hypothesis, the P MA1_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 s uch cavities usually harbor bound water
molecules [11–13]. Furthermore, control calculations repor-
ted here, in which the same approach is applied to the
highest r esolution structures of the proton pump bacterio-
rhodopsin and halorhodopsin, reveal a good correspon-
dence between the positions of identified polar cavities,
water m olecules 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 well-
defined pathway connecting the phosphorylation site in the
extracellular domain to the intracellular side of the mole-
cule. Most of these cavities are in contact with residues
previously shown to affect coupling of proton t ranslocation
to ATP hydrolysis. Some a re also in contact w ith residues
whose r ole in proton transport has as yet not been analyzed.
This per tains in particular to residues in the cytoplasmic
domain which might b e involved in the pathway of proton
entry.
In the current absence o f d etailed 3D d ata, these
suggestions could be tested by mutatagensis e xperiments.
Furthermore, the approach might be a useful preliminary

structure (1EUL.pdb) (Table 1 ), taking into account
the position of aromatic residues a t the boundaries of the
transmembrane segments. It was verified that none of t hese
algorithms included this 3D structure in its learning set. This
is certainly important for t he
DAS
and
TMPRED
algorithms
which rely directly on a datab ase of known structures.
The predictions made for PMA1_NEUCR were com-
pared with experimental d ata on t he insertion i nto micro-
somes of r ecombinant peptides f rom helices M3, M5, M7,
M8 and M10 [19,20] (Table 2).
In the case of ATC1_RABIT, combining t he predictions
from the different algorithms yielded accurate predictions
for n ine out of 10 transmembrane s egments. 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 a verage transmembrane topology for PMA1_NEU
CR, presented in Table 2, c ontains 10 transmembrane
Table 1. Topological predictions and average topological model for ATC1_RABIT. Comparison with the t opology of the crystal structure.
DAS
(1.7)
HMMTOP PHDHTM TMHMM TMPRED
Average
M1 60–76 60–78 59–76 60–78 60–78 60–77 57–77
M2 88–104 85–106 83–106 85–107 85–107 85–106 88–107
– 207–228 – – 207–226 – –

2+
-ATPase (ATCI_RABIT) were retrieved from the
Swissprot database [22].
Obtaining a correct sequence alignment is the corner stone
of success in all homology modeling procedures. Here two
different algorithms were used to align the two sequences.
CLUSTAL W
[23] was u sed t o g enerate 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 m ethods, the Blosum62 substitution matrix
was used and the open gap and extension penalties were 1 2–
2for
CLUSTALW
and 12–4 for
SIM
.
PHDsec [25] was used to g enerate 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 s imilarity

Consequently, the raw global a lignment 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 c oncerned very short segments s epar-
ated by large gaps. Information on the predicted trans-
membrane 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 con-
sistent with th e
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 man ually by aligning the corresponding transmem-
brane segments of both enzymes while maximizing sequence
identity.
As mentioned above, the prediction for the M 8 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 r egulatory domain in
PMA1_NEUCR, which showed that the 897–920 s egment
had a cytoplasmic l ocation [27]. In plant H
+
-ATPases,
where an equivalent domain is also located after M10, this
domain was suggested to intera ct directly with the enzy me

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. 1 with a
comparison between t he secondary str ucture predictions for
PMA1_NEUCR sequence and the a ssigned secondary
structure of the 3D model. Although this secondary
structure p rediction was not used in the alignment p art o f
the modeling procedure, it is presented here to show that
in the cytosolic domains, the secondary structu re p redic-
tion and the secondary structure resulting from the
modeling p rocedure a re remarkably consistent. Position
of PMA1_NEUCR and A TC1_RABIT transmembrane
segments are indicated as shaded colored boxes.
Model building. Using the sequence alignment displayed i n
Fig. 1 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 w ith the loop database module
of
DEEPVIEW
.
Model quality assessment and refinement
The quality of the model was assessed using t he
WHATIF

followed by 300 steps o f conjugate g radient optimizations.
This led to a significant drop in the unfavorable nonbonded
contacts, while producing only very minor displacements o f
the atomic coordinates.
Identification of internal cavities
Internal cavities were identified from the atomic coordi-
nates of the PMA1_NEUCR model, ATC1_RABIT
(1EUL.pdb), bacteriorhodopsin (1C3W.pdb), halorhodop-
sin (1E12.pdb),
L
-2-haloacid dehalogenase (1JUD.pdb) and
phosphoserine phosphatase (1F5S.pdb) using the surface
modul e of
DEEPVIEW
3.7 and the program
SURVOL
[34].
In both programs the computed cavities were delimited by
the m olecular 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 transmem-
brane segments M4, M5, M6 and M8. The crystal structure
of ATC1_RABIT reveals how these residues assemble to
form a binding pocket surrounding two Ca

[39]. In plant H
+
-ATPases (Arabidhopsis thaliana), how-
ever, the conserved Asp residue does not seem to be
involved in a salt bridge a nd 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 1 of A TC1_RABIT, only S er699 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 r esidues, 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 a long segment M 4 of
yeast H
+
-ATPase showed that replacement of Ile331 and
Val334 had little o r n o effect on A TP-dependent proton
transport [41], not inconsistent with the fact t hat the
mutations do not change the nature of the backbone.
Replacement of Ile332 or V al336 resulted, however, in a
coupled mutant enzyme displaying altered kinetics consis-
tent with a slow down of the E
1

The chemiosmotic model for PMA1_NEUCR. In the
P-type proton pumps, the origin of the transported proton
as well as the proton e ntry pathway is still unknown. The s o-
called Ôchemiosmotic modelÕ for PMA1_NEUCR, based
largely on biochemical data, m akes an interesting p roposal
concerning the initiation site f or 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 o f the model are as follows: The first
step is a covalent phosphoryl-transfer reaction from the
MgATP m olecule to the strictly conserved Asp residue
(Asp378), as shown by radioactively labeled ATP [42,43].
The next step is dephosphorylation of the aspartyl-
phosphate group with subsequent release of phosphate
at the cytoplasmic side o f the enzyme. This reaction
involves a water molecule whose oxygen atom p romotes
disruption o f the covalent bond between the conserved
Asp378 residue and P
i
[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 w ith other protein groups and with one
another [11–13]. Such cavities could therefore represent loci

(Thr46, Tyr57, Arg82, Asp85, Asp96, Glu194, Glu204,
Glu205, Asp212 and Lys216) are believed to be involved in
the network of the hydrogen-bonded residues a nd 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 A
˚
3
.Mostof
them are located along the l ongitudinal axis o f the protein as
seen in Fig. 4. Along this axis, two main groups of cavities
can be distinguished. The first is located just below the
cytoplasmic s ite of M gATP hydrolysis. The second group,
located in the membrane domain, almost connects the
region homologous to the ion-binding site to the extra-
cytoplasmic 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 w ith the semi-empirical chemiosm otic model for
PMA1_NEUCR [49].
In order to verify this suggestion, we listed all the residues

+
-ATPase (Eur. J. Biochem. 269) 5251
defective in the E1-E2 conformational change. Here, the
Val336Ala mutant displaye d k inetic properties consistent
with a decrease of the transport-linked E1P–E2P transition
step [41]. As s een above, Val336 corresponds to Glu309 of
ATC1_RABIT that contributes d irectly to calcium b inding
site 2 (Fig. 2).
The following long cavity (cavity 9) is located between
helices M4, M5, M6 and M8 again in the r egion
corresponding to the ion-binding site. A conserved residue,
Tyr694, is making contact w ith this c avity. The Tyr694 to
Ala mutant strongly decreased ATPase activity, while the
Tyr694 to Gly m utant displayed a strong resistance towards
inhibition by vanadate. Interestingly, Tyr694Ala mutant
resulted in a presumably uncoupled enzyme, although the
low r ate of ATP hydrolysis prevented a detailed a nalysis of
the coupling ratio. In ATC1_RABIT, mutation of the
corresponding residue (Tyr763), clearly led to an uncoupled
enzyme unable to transport Ca
2+
ions [53]. Another residue
in contact w ith cavity 9 is Ser699 that was found to be
involved in proton translocation (see a bove).
Two other cavitie s are observed between M6, M8 a nd M9
that are in t he 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 th erefore considered as less reliable than in other
parts of the model. It i s nonetheless of in terest that residue

in (B) is rotated by nearly 90 ° with respect to
(A) (the NH
2
-terminus is omitted for clarity).
Cavities are colored by atom type (red stand s
for O, blue for N and yello w for S). The
horizontal black lines delineate the approxi-
mate membrane position. The catalytic Asp
(Asp378) appears i n red.
5252 O. Radresa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Table 3. List of the residues in contact with the cavities 1 to 9. 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 Mutation Expression Coupling Kinetic E1–E2 ATPase activity Reference
Cavity 1
Rossman fold K615 – – – –
M631 – – – –
T632 – – – –
G633 – – – –
D638 N Lethality [60]
S641 – – – –
L642 – – – –
Cavity 2
Rossman fold C376 A 104% ND ND 94% [61]
S377 A 103% ND ND 15% [54]
D378 N, S – – Misfolded –
E – – Misfolded – [62]
L557
K615
–
–

Cavity 5
Rossman fold L369 A 86% (a) Altered 12% [55]
A630 – – – –
T647 – – – –
I649 – – – –
I664 – – – –
F666 – – – –
Stalk M5 I674 – – – –
A677 – – – –
L678 – – – –
Cavity 6
Stalk M4
M346 A 96% Normal Altered 55% [41]
G349 A 37% ND Normal 23% [41]
A350 S 85% Normal Normal 123% [41]
L353 A 44% ND Normal 36% [41]
V360 A 84% Normal Altered 45% [41]
I366 A 54% Normal Altered 77% [55]
Stalk M5 L369 A 86% ND Altered 12% [55]
A677 – – – –
T680 – – – –
S681 – – – –
Ó FEBS 2002 3D modelling of Neurospora H
+
-ATPase (Eur. J. Biochem. 269) 5253
We thus see that the pathway outlined by the c avities
numbered 7–10 connects the bottom of the phosphorylation
site organized in a Rossman fold to the extracytoplasmic
moiety. Furthermore, a ll these cavities, with the exception o f
cavity 7, are lined by one or more residues previously shown

An apparent pathway by which a proton might be
internalized from the cytosol to reach cavity 6 is through
cavities 1–5. Indeed, polar residues a re lining these cavities,
consistent with the idea t hat they m ight harbor a water-
mediated H-bond network fostering proton transfer.
However, a potential problem in validating this p roposal
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 linin g cavities
1–5 are found in the vicinity of t he ÔAMTGDGVNDAP640Õ
motif, a r egion that h as as yet not been thoroughly
investigated. T he residues identified in this region (Table 3)
might thus c onstitute su itab le targets f or sit e-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 a vailable from mutagenesis studies, positions of
the cavities l ocated in our model were compared w ith those
Table 3. (Contin ued).
Region Mutation Expression Coupling Kinetic E1–E2 ATPase activity Reference
Cavity 7
TM3 V289 F ND ND Altered revertant
of S368F
103% [50]
L67 I293 – – – –
W756 – – – –
G757 – – – –
Cavity 8
TM4 P335 A 80% (a) Normal 22% [41]

the HAD superfamily (Fig. 5).
We used the high-resolution structures of the
L
-2-haloacid
dehalogenase (1JUD) and o f 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% an d 66% of th e
Ca comprising the Rossman fold displayed an root mean
squared d eviation of less than 1.85 A
˚
with the homologous
domain of P MA1_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 crystallogra-
phic water molecule s were identified in these structures u sing
the d escribed procedure. Quite strikingly, in both cases, the
structures e xhibit a single internal polar cavity in the vicinity
of the conserved catalytic Asp. This cavity contains two to
three crystallographic w ater molecules. In stark contrast, the
Rossman fold of PMA1_NEUCR contains as many as six
polar cavities, w hich link the phosphorylated Asp378 all
through the middle of t he a/b Rossmanfold,downtothe
beginning of the transmembrane domain. The presence of
this string o f polar cavities in P MA1_NEUCR Rossman
fold, the most accurate part of our model, and its

sent buried crystallographic water molecules;
blue dots represent crystallographic water
molecules lying ou tside the su rfac e envelope o f
the enzymes. Cavities are colored by atom
type (red stands for O, blue for N and yellow
for S).
Ó FEBS 2002 3D modelling of Neurospora H
+
-ATPase (Eur. J. Biochem. 269) 5255
possesses some specific chemical and structural f eatures
enabling them to fulfill their specialized transport function.
In the second part of this work, the model of
PMA1_NEUCR was used to p redict a proton transport
pathway that would start near the phosphorylation site at
the c ytoplasmic domain, pass through the transmembrane
region and e nd 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 i n the high-resoluti on structure of bacte-
riorhodopsin, without any prior info rmation.
Some of the residues lining t he identified cavities in
PMA1_NEUCR were shown previously to be involved in
proton transfer by mutagenesis e xperiments, whereas the
role of others has as y et 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 phos-
phorylation site, which is t he most reliable portion of our
3D model. Given the over all low sequence similarity

this domain. Likewise, the secondary structure o f the
phorphorylation domain deduced from the model built
here using the Ca
2+
-ATPaseastemplateandthat
determined directly from the a mino acid sequence of the
H
+
-ATPase, are a lso in excellent agreemen t. All this
indicates t hat the ter tiary 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, warrant-
ing some confidence in the proposed proton entry
pathway, which is mostly located in this domain. A good
indication that the delineated pathway might be relevan t
to function is our finding that a s imilar pathway cannot
be traced in the structurally related soluble members of
the HAD super-family, which unlike PMA1_NEUCR do
not mediate proton translocation.
The proton transport pathway proposed here is consis-
tent with the semiempirical chemiosmotic model for p roton
transport i n 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 do not believe that such ion is
the transported species. We favor instead the well docu-
mented mechanism of proton conduction through a
network o f h ydrogen-bonded internal water molecules

proton translocation. J. Biol. Chem. 261, 7466–7471.
4. Auer, M., Scarborough, G.A. & Kuhlbrandt, W. (1998) Three-
dimensional map of the plasma membrane H
+
-ATPase in the
open conformation. Nature 392, 840–843.
5. 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.
6. Scarborough, G.A. (1999) Structure and function of the P-type
ATPases. Curr. Opin. Cell. Biol. 11, 517–522.
7. Stokes, D.L., Auer, M ., Zhang, P. & Kuhlbrandt, W. (1999)
Comparison of H
+
-ATPase and Ca2+-ATPase suggests that a
large c onformationa l change initiates P-type ion pump re action
cycles. Curr. Biol. 9, 672–679.
8. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000)
Crystal s tructure of th e calcium pump of sarco plasmic reticulum
at 2.6 A
˚
resolution. Nature 405, 647–655.
9. Bukrinsky, J.T., Buch-Pedersen, M.J., Larsen, S. & P almgren,
M.G. (2001) A putative proton-binding site of plasma membrane
H
+
-ATPase identified through h omology modelling. FEBS Lett.
494, 6–10.

Eds), pp. 175–182. AAAI Press, Menlo Park, CA.
18. Hofmann, K. & Stoffel, W. (1993) T Mbase – a database of
membrane spanning prot eins segme nts. Biol. Chem. Hoppe-Seyler.
347, 166–171.
19. Lin, J. & Addison, R. (1994) Topology o f the Neurospora plasma
membrane H
+
-ATPase. Localization of a transmembrane seg-
ment. J. Biol. Chem. 269, 3887–3890.
20. Lin, J. & Addison, R. (1995) The membrane topology of the
carboxyl-terminal third o f the Neurospora plasma me mbrane H
+
-
ATPase. J. Biol. Chem. 270, 6942 –6948.
21. Cuff, J.A., Clamp, M.E., S iddiqui, A.S., F inlay, M. & Barton, G.J.
(1998) Jpred: a c onsensus secondary structure prediction server.
Bioinformatics 14, 892–893.
22. Appel, R.D., Bairoch, A. & Hochstrasser, D.F. (1994) A new
generation of information retrieval tools for biologists: the
example of t he ExPASy WWW server. Trends Biochem. Sci. 19,
258–260.
23. Thompson, J.D., Higgins, D.G. & G ibson, T.J. (1994) CLUS TAL
W: improving the sensitivity of progressive multiple sequence
alignment t hrough sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22,
4673–4680.
24. Huang, X. & Miller, W. (1991) A time-efficient, linear-space l ocal
similarity algorithm. Adv. Appl. Math. 12, 337–357.
25. Rost, B. & Sande r, C. (1994) Combining evolutionary info rmation
and neural networks to predict protein secondary structure.

(1996) Biomolecular Simulation: t he GROMOS96 Manual
and User Guide. Vdf Hochschulverlag AG an der ETH Zu
¨
rich,
pp. 1–1042. Zurich, Switzerland.
34. Pontius, J ., Richelle, J. & Wodak, S.J. (1996) Quality assessment
of protein 3D structures using standard atomic volu mes. J. Mol.
Biol. 264, 121–136.
35. Clarke, D.M., Loo, T.W., Inesi, G. & MacLennan, D.H. (1989)
Location of high affinity Ca
2+
-binding sites within the predicted
transmembrane domain of the sarcoplasmic reticulum Ca
2+
-
ATPase. Nature 339, 476–478.
36. Jorgensen, P.L. & Pedersen, P.A. (2001) Structure–function
relationships o f N a
+
,K
+
,ATP,orMg
2+
binding and
energy transduction in Na,K-ATPase. Biophys. Biochim. Acta
1505, 57–74.
37. Dutra, M.B., Ambesi, A . & Sl ayman, C.W. ( 1998) Structure-
function relationship in membrane segment five of the yeast
PMA1 H
+

-ATPase as a beta-aspartyl phosphate. J. Biol. Chem. 256,
10724–10730.
43. Dame, J.B. & Scarborough, G.A. (1980) I dentifi cation of the
hydrolytic moiety of the Neurospora plasma membrane H
+
-AT-
Pase and demonstration of a p hosphoryl-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 Schizosaccharo-
myces pombe. J. Biol. Chem. 257, 12509–12516.
45. Kandori, H. (2000) Role of internal water molecules in bacterio-
rhodopsin. 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 mecha-
nisms o f the cation -motive ATPases. Ann. NY Acad. Sci. 402, 99–

plasmic reticulum. J. Biol. Chem. 270, 908–914.
54. DeWitt, N .D., Tourinho dos Santos, C.F., Allen, K.E. & Slay-
man, C.W. (1998) Phosphorylation region of the yeast plasm a-
membrane H
+
-ATPase. Role in protein folding and b iogenesis.
J. Biol. Chem. 273, 2 1744–21751.
55. Ambesi, A., Miranda, M., Allen, K.E. & Slayman, C.W. (2000)
Stalk segment 4 of t he ye ast p lasma membrane H
+
-ATPase.
Mutational evidence for a role in the E1–E2 co nformational
change. J. Biol. Chem. 275, 20545–22050.
56. Wang, W., Kim, R., Jancarik, J., Yokota, H. & K im, 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 bac-
terial haloacid dehalogenases defines a large superfamily of
hydrolases with diverse specificity. A pplication of an i terative
approach to database search. J. Mol. Biol. 244, 125–132.
58. Aravind, L., Galperin, M.Y. & Koonin, E.V. ( 1998) The catalytic
domain of the P-type ATPase has the haloacid dehaloge nase fold.
Trends Biol. Sci. 23, 127–129.
59. Stokes, D.L. & Green, N.M. (2000) Mo deling a dehalogenase fold
into th e 8-A
˚
density map for Ca