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

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

Neurospora crassa plasma membrane H
+
-ATPase, using as
template the 2 .6 A
˚
crystal structure of rabbit sarcoplasmic
reticulum C a
2+
-ATPase [Toyoshima, C., Nakasako, M.,
Nomura, H. & Ogawa, H. (2000) Nature 405, 647–655].
Comparison of the two calcium-binding s ites in the crystal
structure of Ca
2+
-ATPase with t he equivalent region in the
H
+
-ATPase m odel shows that t he latter is devoid of most of
the negatively c harged groups required to bind the cations,
suggesting a different role for this region. Using the built
model, a pathway for proton t ransport is t hen 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 pu mp bacterio-
rhodopsin. This revealed a striking correspondence between
the positions of internal polar cavities, those of crystallo-
graphic w ater molecu les a nd, in the case of bacteriorho-
dopsin, the residues mediating proton translocation. In our
H
+

-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-

spora 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 o f ionic substrates including
charged amino phospholipids [1]. PMA1_NEUCR compri-
ses 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 m onomeric 100 k Da
polypeptide chain, it transports ions outs ide the cell in a n
electrogenic way using energy from M gATP 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 Ca
2+
-ATPaseofrabbitsarcoplasmic
reticulum ( referenced hereafter as ATC1_RABIT), have
been determined at 8 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 tran smembrane
segments. Comparison of the two low-resolution models,
which are believed to represent different conformational
states, r evealed that they displayed strikingly similar pack-
ing of their respective 10 transmembrane segments whereas
their c ytoplasmic domains appeared too different to allow
direct superimposition [6,7].

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:
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

8A
˚
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 l oop responsible for ATP
hydrolysis were built for p lant and yeast H
+
-ATPases on
thebasisoftheACT1_RABITcrystalstructure[9].From
these m odels is was proposed that proton transport in the
H
+
-ATPases is mediated through specific binding of a
hydronium ion at a site structurally eq uivalent to the second
calcium binding site in ACT1_RABIT.
In this work, we combine homology modeling t echniques
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 t ransport pathway. This hypo-
thesis is based on the assumption that as in the case of t he
well-known b acteriorhodopsin proton pump, H
+
ions
would be t he relevant transported species, w ith t heir
transport mediated by one or more acidic side chains of

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

tool for identifying putative proton pathways in other
proton-transporting enzymes for which structural data a re
available.
MATERIALS AND METHODS
Building the 3D model of PMA1_NEUCR
In our approach, the model of PMA1_NEUCR was built
using the A TC1_RABIT crystal structure as template by
combining transmembrane topology predictions with stand-
ard 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 transmem-
brane topology of PMA1_NEUCR was predicted from
the amino acid sequence by averaging the results of five
different predictive a lgorithms:
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 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 – –

M3 263–276 261–279 259–276 – 260–279 261–278 256–279
M4 294–308 286–307 295–314 – 292–314 292–311 288–312
M5 769–784 772–793 773–794 – 769–790 771–790 760–778
M6– –––––789–809
M7 832–851 838–859 833–850 837–859 839–855 835–855 832–854
M8 – – – – 896–917 896–917 891–914
M9 930–954 – 932–949 928–950 928–950 930–951 932–952
M10 967–980 967–980 – – – 967–980 967–986
Ó FEBS 2002 3D modelling of Neurospora H
+
-ATPase (Eur. J. Biochem. 269) 5247
segments in agreement with the 8 A
˚
electron density map
and w ith experimental data on recombinant peptides. It is
worth noting at this point that these data do not provide
information on the precise boun daries 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 o f the M8 helix in
the native protein [20], the
TMHMM
predictions for M 8
were used.
Sequence alignment. The sequences of Neurospora crassa
H
+
-ATPase (PMA1_NEUCR) and rabbit SERCA1a/
Ca

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

between the t arget 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 cytop lasmic
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 do not interfere w ith the core domain.
Aside from t his gapped c ytoplasmic loop, sequence
identity in this domain exceeded 30% making this part of
the alignment m ore straightforward. We could furthermore
verify that this sequence alignment was consistent wit h the
alignment between the secondary structures predicted solely
from the sequence o f PMA1_NEUCR and those assigned
in the equivalent r egions 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 followin g the l ast transmembrane
segment M 10.

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

active site [28]. In the ATC1_RABIT template, the active
site is located in the large c ytoplasmic loop between M4 and
M5, almost 45 A
˚
away from the end of M10. These
considerations impose some constraints on the
Table 2. Topological predictions and average topological model for PMA1_NEUCR. Comparison with available data on recombinant peptides.
Predictive algorithms
Recombinant
peptidesDAS (1.7) HMMTOP PHDhtm TMHMM TMPRED Average
M1 116–126 113–137 117–134 – 116–136 116–133 –
M2 144–159 145–169 142–159 – 141–160 143–162 –
M3 291–312 289–313 297–314 292–314 292–310 292–313 292–314
M4 322–348 327–351 325–343 327–349 327–354 326–349 –
664–674 – – – 660–678 – –
M5 691–714 689–713 697–721 691–713 688–713 691–715 688–713
M6 717–734 721–741 – 720–738 721–738 720–738 721–738
M7 757–774 755–779 758–775 757–775 755–774 756–775 755–779
M8 – – – 796–814 – 796–814 807–826
M9 828–843 822–846 826–843 829–849 827–847 826–846 827–847
M10 854–877 854–878 859–876 860–878 860–877 856–878 854–878
5248 O. Radresa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
PMA1_NEUCR model in this region. In particular, the
regulatory domain of PMA1_NEUCR should be able t o
reach across the active site. If we believe that it adopts a n
a-helical structure a s indicated by t he prediction (Fig. 1),
then the corresponding helix would have to be of at least 30
residues l ong. This implies i n turn that t he 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. 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

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
Fig. 1. Sequence alignment of PMA1_NEUCR and ATC1_RABIT. PMA1_NEUCR PHD: secondary structure prediction o f P MA1_NEUCR using
thePHDsecalgorithm.PMA1_NEUCR DSSP: seco ndary struct ure of the mode l structure a ssigned with the DS SP algorit hm (arrows: b-sheets;
helices: a-helices). Shaded colored box: tran smembrane d omains of ATC1_RABIT and PMA1_NEUCR. Shaded orange box: PMA1_NEUCR
transmembrane segments built with the t opology-based alignment. Numbers below the sequences refer to th e residues in direct contact with the
cavities 1 t o 9 in PMA1_NEUCR; italic font is used when a mut ation has been reported. Residues appearing in white over a red background are
identical. Residues appearing in red over a white background are homologous.
Ó FEBS 2002 3D modelling of Neurospora H
+
-ATPase (Eur. J. Biochem. 269) 5249
were of similar quality, indicating that care was taken in
both the homology modeling and X-ray refinement proce-
dures to optimize these parameters.
However, significant differences were observed in t he
number o f close nonbonded contacts. Those were higher in
the constructed model of PMA1_NEUCR than for the
crystal s tructure of the r abbit enzyme. To relieve this strain
the model was subjected to two short energy minimization
runs with
GROMOS
96 [33] using t he
GROMOS
43B1 f orce
field. These runs involved 200 steps of s teepest descent

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

2+
ions. A
comparison of this bind ing pocket with the corresponding
domain of our 3D m odel of PMA1_NEUCR is s hown i n
Fig. 2.
Ion binding site I. Five residues form the first Ca
2+
binding site. The calcium ion is bound to the side-chain
oxygen of Asn768 and Glu771 on M5, of Thr799 a nd
Asp800 on M6 and G lu908 on M 8. All the oxygens are
arranged in roughly the same plane except for the side chain
of Glu771, which is located below.
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 mutagen-
esis along M5 [37] showed that Ser699 is probably involved
in proton transport, although it is not essential to the
coupling mechanism. Substitution of Le u702 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 en zyme, arrested in the endoplasmic
reticulum [41]. Nevertheless, double m utants in which both
charged A rg695 and Asp730 were inversed or r eplaced by
Ala reverted to a fully functional enzyme. These observa-
tions 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), 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

P–E
2
P transition step
believed to b e coupled to the charge transfer reaction.
Finally, Asp730 c orresponding to Asp800 in ATC1_RABIT
was shown to be involved in a salt bridge with Arg695
precluding direct participation i n proton t ransport, as
discussed above.
Fig. 2. Comparison of ATC1_RABIT (A) and
PMA1_NEUCR (B) ion-binding sites regions.
Calcium ions in A TC1_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 b y the dotted line.
5250 O. Radresa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In conclusion, comparisons of the residues involved i n
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 r egion 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 i n ATC1_RABIT m ight 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 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

where water molecules could form H-bond networks that
foster proton conduction [46,48]. On the basis of this
hypothesis, we s et 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 i n
bacteriorhodopsin [45]. To lend support to our hypothesis,
we therefore applied the same approach to the high-resolu-
tion 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 t he buried crystallographic water mole cules
are located in internal polar cavities, as ill ustrated in
Fig. 3A,B [46,47]. In the case o f t he bacteriorhodopsin
proton pump, eight s uch cavities have been identified. The
15 polar residues t hat line these cavities are Thr5, Arg7,
Thr46, Tyr57, Tyr79, Arg82, Tyr83, Asp96, Ser193, Glu194,
Glu204, Tyr205, Asp212, and L ys216. 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 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

lining these cavities and compared them to those shown to
affect the proton t ransport reac tion by mutage nesis experi-
ments. The cavity-lining residues number 54 i n all, of which
22 are identical to those in A TC1_RABIT (see alignment of
Fig. 1).
Table 3 lists the 5 4 residues, together with the cavity they
contact, and the effect of mutations on the kinetic properties
and the coupling r atio between ATPase a ctivity and proton
transport. These residues are numbered on t he sequence
alignment o f F ig. 1, in italic fonts when a mutation has b een
reported or in regular font when they h ave 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 i n 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
Ca
2+
binding sites by site-directed mutagenesis [51,52].
The next c avity 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
Ó FEBS 2002 3D modelling of Neurospora H

+
-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

Glu805 corresponding to Glu803 in the yeast H
+
-ATPase
is found close to cavity number 10, a s mall internal cavity
that app ears as nonpolar in our m odel. 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 i s partially accessible to the solvent and m ay
therefore act as a gate for the possible exit of a water-
coordinated proton occurring at the extra-cytoplasmic end
of the proton transfer path.
Fig. 3. Position of internal polar cavities and
crystallographic water molecules in the high
resolution crystal structures of bacteriorhodop-
sin (A) and halorhodopsin (B). Red spheres
represent buried crystallographic water mole-
cules; blue dots represent crystallographic
water molecules lying outside the surface
envelope of the e nzymes. Cavities are colored
by atom type (re d stan ds for O , blu e for N a nd
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. 4. Position of internal polar cavities in
PMA1_NEUCR model. (A) a nd (B) views and
numbering of internal polar cavitie s. The vie w

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
–
–

–
–
–
–
–
–
M631 – – – –
T632 – – – –
Cavity 3
Rossman fold T382 A 14% Misfolded ND [54]
T632 – – – –
G633 – – – –
I649 – – – –
A650 – – – –
V651 – – – –
F666 – – – –
P669 – – – –
G670 – – – –
L671 – – – –
I674 – – – –
Cavity 4
Rossman fold L375 A 31% Misfolded 29% [54]
C376 A 104% ND ND 94% [61]
S377 A 103% ND ND 15% [54]
A630 – – – –
M631 – – – –
T632 – – – –
I649 – – – –
L671 – – – –
I674 – – – –

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

to be involved in the p roton translocation step.
(b) Internal polar cavities in the cytoplasmic domain.
The cavities identified in this domain trace a c lear path that
starts in th e cent er of t he 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 a cids adjacent to
the phosphorylated Asp [54], limited information is avail-
able from mutagenesis studies on residues involved in
proton translocation in the cytoplasmic phosphorylation
site.
At the bottom of the cytoplasmic domain, cavity number
6 i s in contact with two residues of interest f or the proton
translocation step: Met346 and I366. Mutations of either
Met346 or Ile366 to Ala result in a reduced K
m
for MgATP,
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 E
1
P–
E
2
P 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 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]

V336 A 108% Normal Altered 96% [41]
TM5 Y691 A 92% ND Altered 0% [37]
R695 A 14% Misfolded salt bridge
with D730 revertant
[37]
[39]
D of D730R [39]
TM6 D730 N, V < 10% Misfolded Unactive [39]
E < 10% ND ND 15% [38]
L734 – – – –
Cavity 9
TM4 I332 A 71% Normal Altered 32% [41]
TM5 Y694 A 40% (a) Altered 22% [37]
R695 A 14% Misfolded [37]
L698 A 90% Normal Normal 70% [37]
TM5 S699 A 90% (a) Normal 8% [37]
C 79% Normal pH shift 16% [37]
T 94% Normal Normal 96% [37]
H701 A 15% ND ND Unactive [37]
L702 A 39% ND Altered 42% [37]
I725 – – – –
TM6 A726 – – – –
A729 – – – –
D730 N, V < 10% Misfolded Unactive [39]
E < 10% ND ND 15% [38]
L734 – – – –
TM8 F810 – – – –
5254 O. Radresa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 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

conspicuous absence in the otherwise close structural
relatives from the soluble members of the HAD family,
point to a possible functional role o f these cavities in
PMA1_NEUCR.
CONCLUSION
In this study, we built a c omplete a tomic m odel of
Neurospora crassa plasma membrane H
+
-ATPase
(PMA1_NEUCR) using the high-resolution 3D structure
of the rabbit sarcoplasmic reticulum Ca
2+
-ATPase
(ATC1_RABIT) as a template and combining transmem-
brane topology predictions with classical homology mode-
ling techniques in regions of low sequence s imilarity.
In the first part of this work, t he comparison of the ion-
binding site of ATC1_RABIT with the homologous domain
in PMA1_NEUCR revealed that this doma in is not
conserved in both enzymes. This suggests that although
the P-type ATPases are widely assumed to share a common
mechanism of a ction, each group of proteins probably
Fig. 5. 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 R oss-
man fold appears in red. Red s pheres repre-

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

between the H
+
-andCa
2+
-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 w ith
limited accuracy. T his 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 w here we locate the proposed
proton entry p athway is a much more reliable portion of our
model, as the s equences of the eukaryotic P-type ATPases
are essentially conserved in t his domain. Moreover, we
illustrated that the fold of this domain is remarkably
conserved between the C a
2+
-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 p redicted
secondary structures for s everal P-type ATPases including
the H
+
-ATPase are also remarkably well conserved in

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

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 experi-
mental 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 p rovide important clues on the
molecular mechanism u sed by t hese complex proton pumps.
ACKNOWLEDGMENTS
O. R . is recipient of the ÔFonds pour la Fo rmation a
`
la Recherche d ans
lÕIndustrie et dans l’Agriculture’ (Belgium). E. G. is Research Director
of the ÔFonds National de la Recherche S cientifiqueÕ (Belgium). We
thank th e ÔCommunaute
´
Franc¸ aise de Belgique: Actions de Recherche
Concerte
´
esÕ for financial support.
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