Báo cáo khoa học: Crystal structure of the tetrameric inositol 1-phosphate phosphatase (TM1415) from the hyperthermophile, Thermotoga maritima docx

Crystal structure of the tetrameric inositol 1-phosphate
phosphatase (TM1415) from the hyperthermophile,
Thermotoga maritima
Kimberly A. Stieglitz
1
, Mary F. Roberts
1
, Weizhong Li
2
and Boguslaw Stec
2
1 Merkert Chemistry Center, Boston College, Chestnut Hill, MA, USA
2 The Burnham Institute for Medical Research, La Jolla, CA, USA
The extended inositol monophosphatase (IMPase) ⁄
fructose-1,6-bisphosphatase (FBPase) family provides
an interesting case in which the changes in 3D archi-
tecture and structural flexibility can be linked to
increased specialization and emergence of new func-
tion. This structural family was identified at the begin-
ning of the 1990s [1,2]. Since then, the rapid progress
in genome sequencing has added many new members.
At present, the family contains around 1000 sequences,
classified in three Pfam5 subfamilies, of which  600
are in the IMPase subfamily. Despite having the same
architectural features at the single monomer level, the
proteins in this family show increasingly complex
oligomeric organization. The inositol polyphosphate
1-phosphatases and 3¢-phosphoadenosine-5¢-phosphate
phosphatases are monomeric. Most of the IMPases are
dimeric, although the Escherichia coli enzyme (also
known as SuhB) can be monomeric as well as dimeric

[3]. All of the eukaryotic FBPases are tetrameric, and
some are allosterically regulated by AMP [4] or oxida-
tion of disulfides [5].
The IMPases in higher eukaryotes are involved in
secondary messenger signaling by regenerating the
myo-inositol pool, whereas the bacterial and archaeal
counterparts may be involved in osmolyte synthesis, as
has been postulated for the IMPase from Thermoto-
ga maritima (TM1415) [6]. Members of this family
show reactivity towards several substrates, including
poly phosphorylated inositol species and phosphorylat-
ed nucleotides. A few IMPase enzymes from hyper-
thermophilic organisms also catalyze the hydrolysis of
the 1-phosphate of FBP; these have specific FBPase, as
well as IMPase, activities [7]. We have previously
solved two structures from this superfamily – Metha-
nocaldoccocus jannashii IMPase (MJ0109) [7,8] and
Archaeoglobus fulgidus IMPase (AF2372) [9] – and in
Keywords
fructose-1, 6-bisphosphatase; inositol
monophosphatase; protein folding;
thermophile; Thermotoga maritima
Correspondence
B. Stec, The Burnham Institute, 10901 N.
Torrey Pines Road, La Jolla, CA 92037, USA
Fax: +1 858 7139948
Tel: +1 858 7955257
E-mail:
(Received 15 December 2006, revised 5
March 2007, accepted 8 March 2007)

doi:10.1111/j.1742-4658.2007.05779.x
The structure of the first tetrameric inositol monophosphatase (IMPase)
has been solved. This enzyme, from the eubacterium Thermotoga maritima,
similarly to its archaeal homologs exhibits dual specificity with both
IMPase and fructose-1,6-bisphosphatase activities. The tetrameric structure
of this unregulated enzyme is similar, in its quaternary assembly, to the
allosterically regulated tetramer of fru ctose-1,6-bi sphosph atase. The indi vidual
dimers are similar to the human IMPase. Additionally, the structures of
two crystal forms of IMPase show significant differences. In the first crystal
form, the tetrameric structure is symmetrical, with the active site loop in
each subunit folded into a b-hairpin conformation. The second form is
asymmetrical and shows an unusual structural change. Two of the subunits
have the active site loop folded into a b-hairpin structure, whereas in the
remaining two subunits the same loop adopts an a-helical conformation.
Abbreviations
AF2372, IMPase from Archaeoglobus fulgidus; FBPase, fructose-1,6-bisphosphatase; IMPase, inositol 1-phosphate phosphatase;
TM1415, IMPase from Thermotoga maritima; MJ0109, IMPase from Methanocaldococcus jannaschii.
FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS 2461
this report we present the structure for TM1415, the
IMPase ⁄ FBPase from T. maritima.
The proteins in the family share a common mono-
mer architecture and have common principles of chem-
ical reactivity. A three metal ion-assisted catalytic
mechanism was proposed to function in the entire fam-
ily [8,10]. It has also been suggested that the enzymes
might have a common origin in a postulated ancient
cyclitol phosphatase [7]. One of the common elements
of this architecture is a loop that closes the active site
once the substrate binds. Many of the eukaryotic
members of this family (including Homo sapiens

IMPase, inositol polyphosphate 1-phosphatase, and
FBPase) are inhibited by submillimolar concentrations
of lithium ions [11–13]. Li
+
therapy is an important
remedy against common mental disorders [14], and
inhibition of some of the human IMPase family
enzymes is believed to be responsible for these thera-
peutic effects. We have proposed that the mobile loop
is the determinant of sensitivity to Li
+
[9].
In this report we provide an interesting addition to
this already intriguing protein family – the crystal
structure of an IMPase (from the hyperthermophile
T. maritima) that has a tetrameric arrangement and is
also the most active IMPase known [6]. Additionally, a
fragment of this IMPase (TM1415) shows unusual
structural duality. The active site loop (residues 22–42)
comes into the direct lattice contact with itself. In one
subunit it has an a helical conformation, whereas its
partner is in a b conformation. Because this fragment
plays a crucial role in catalytic activity and may also
be involved in Li
+
inhibition, the structural duality
supports the idea by Dunker & Wright [15,16], that
flexible protein fragments might be involved in devel-
oping new functionalities in proteins.
Results and Discussion

Structure solution
TM1415 was expressed in E. coli and purified as des-
cribed previously [6]. The purified enzyme was crystal-
lized in two crystal forms, characterized by space
groups P2
1
and P2
1
2
1
2
1
. The structure was solved by
molecular replacement (molrep) [17]. The structure of
both crystal forms showed a tetrameric arrangement of
this enzyme (Fig. 1). More specifically, both crystal
forms contained a single tetramer in the asymmetric
unit. The corresponding models were refined using cns
[18] at 2.2 and 2.4 A
˚
resolution for forms 1 and 2,
respectively. Refinement resulted in an R-factor of
0.209 and 0.178 (Rfree, 0.278 and 0.267) for forms 1
and 2, respectively (Table 1). Stereochemistry meas-
ures, such as the Ramachandran plot and backbone
and side-chain statistics, were well within the expected
values, as indicated by procheck [19]. The electron
density was very good throughout both models (sup-
plementary Fig. S1) with the exception of loop regions,
and in particular two catalytic loops (22–42,60–72) on

one side of the tetramer in the second crystal form.
The C-terminal region appeared to be much more
mobile, as reflected by increased temperature factors.
The model for crystal form 1 contains the terminal
Lys256, whereas the model for crystal form 2 was ter-
minated at Gly254.
The difference in electron density suggested the pres-
ence of multiple water molecules, as well as a single
Mg
2+
in the active site of crystal form 1 (supplement-
ary Fig. S1). This metal ion is found in a classical
octahedral coordination with Asp79, Glu65 side-chain
carboxyls, the carbonyl of Ile81 and three water mole-
cules as direct ligands. In crystal form 2, the difference
in electron densities suggested the presence of a single
tartaric acid molecule per active site. The tetramer
of crystal form 1 is quite symmetrical, whereas the
tetramer of crystal form 2 is highly asymmetrical
(Table 2). The refined models of both forms contain
 300 water molecules.
Monomer architecture: common family structure
As with other enzymes in this family, including
FBPases, the prote in has a well-preserved l ayered struc-
ture of a and b secondary-structure elements (a-b-a-b-a)
(Fig. 1, left panels), falling into two domains. The
lower domain consists of a layer of two a-helices and
an extensive seven-stranded b-sheet containing the act-
ive site. The lower domain is reminiscent of the AMP-
binding domain of eukaryotic FBPases (supplementary

Fig. S2). The active site is located at the bulge of the
C-terminal end of the third strand with a characteristic
DPXD motif, where both aspartates participate in
metal ion binding. The strand is followed by a two-
turn a-helix that forms a phosphate-binding site. The
second domain has a three-layer motif with two layers
of a-helices flanking the second b-sheet. This domain
corresponds to the FBP-binding domain of FBPases.
The two helices of the first domain are connected by a
mobile active site loop that previously was suggested
to modulate activity by co-ordinating the third metal
ion needed for catalysis [8,10]. This loop was also sug-
gested to be involved in Li
+
inhibition [9]. The average
temperature factors for both domains oscillate below
40 A
˚
2
, with helices being much more ordered than the
connecting loops. (See supplementary Fig. S3 for the
temperature factor profiles of both models.)
Structure of TM1415 IMPase K. A. Stieglitz et al.
2462 FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS
Dimer architecture: similarity to human IMPase
Despite being a bacterial enzyme and having signifi-
cant sequence divergence, the overall architecture of
the dimer is reminiscent of the human IMPase (Fig. 1,
right panel). However, the dimer interface is quite dif-
ferent from the human enzyme and other nonarchaeal

IMPases. The superposition of the second subunit of
the MJ0109 structure (1G0H) after superposition of
the first subunit requires a rotation of  15° along the
longest axis of the dimer in one direction, whereas
superposition onto the second subunit of the human
enzyme (1AWB) requires rotation of  15° in the
opposite direction. The rotation around the axis that
intersects the self-contacting residue, Ile173, is denoted
as the dimer twist-angle. To superpose the second sub-
unit on the structure of the eukaryotic FBPase takes a
30° rotation in the twist direction of the archaeal
enzyme. In comparing the dimer orientation of all
these IMPases, we see that the human IMPase has the
extreme dimer twist-angle that is almost 45° off from
the dimer position in FBPases, whereas the TM1415
and MJ0109 constitute intermediate twists spaced 15°
off each other (central panel of Fig. 1).
Tetramer architecture: similarity to FBPase
TM1415, besides being the most active IMPase
( 100-fold more active than its archaeal, bacterial or
eukaryotic counterparts) [6], is also a very efficient
FBPase [7]. It is notable that the TM1415 structure
represents a tetrameric organization for an IMPase
(Fig. 1) reminiscent of the eukaryotic FBPase organ-
ization. Each tetramer is organized as a dimer of
dimers (Fig. 1). Despite the fact that TM1415 lacks
the additional helix (FBPases have three helices at the
tetramer interface), the helical layers in both enzymes
directly interact. This dimer–dimer interface was
shown to be crucial for the transition of the allosteric

signal in pig kidney FBPase [20]. The rotation around
the axis that relates dimers is termed the tetramer
twist-angle. The TM1415 tetramer, despite having a
Fig. 1. Schematic of the transformation of
TM1415 into the eukaryotic FBPase. The
transformation requires  25° rotation of the
dimers around the tetramer axis and  30°
rotation of the monomers in individual
dimers. In the center there is a schematic
of the mutual relationship of the monomers,
in selected members of the family, as indi-
cated by the twist angle of the dimer.
TM1415 constitutes one of the intermedi-
ates between the human IMPase and
FBPase dimer organization. In the surround-
ing panels, clockwise, starting at the top
left, are the structure of the tetrameric
TM1415, the superposition of TM1415 with
the Homo sapiens (human) IMPase (in gold),
the superposition of Sus scrofa (pig) FBPase
with the MJ0109 (in blue), and the tetramer-
ic pig FBPase. The secondary structure ele-
ments are marked in red (helices) and
yellow (b-sheets).
K. A. Stieglitz et al. Structure of TM1415 IMPase
FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS 2463
similar quaternary arrangement to eukaryotic FBPases,
is more twisted, and the lower dimer requires  25°
rotation to superpose with the corresponding dimer of
the R-state FBPase. It can be compared with an over-

rotated T state (the eukaryotic T state is rotated only
17° away from the R state, Fig. 1) [21]. As indicated in
the central panel of Fig. 1, a rotation of  25° in tetr-
amer twist, and an additional dimer twist in both
dimers of  30°, are required to transform TM1415
into the FBPase.
This nonregulated tetrameric organization of the
T. maritima IMPase ⁄ FBPase might represent an evolu-
tionary intermediate between dimeric IMPases and the
allosterically regulated tetrameric eukaryotic FBPases.
As a reminder, not all eukaryotic FBPases are alloster-
ically regulated by AMP (e.g. chloroplast FBPase) [5].
However, the quaternary structures of these IMPase
and FBPases suggest that quaternary organization may
be critical in creating regulatory abilities.
Crystal packing
Both crystal forms of TM1415 have a tetramer present
in the asymmetric unit with similar packing of individ-
ual tetramers. However, in crystal form 2, the packing
is not as tight as in crystal form 1. In crystal form 1
(P2
1
), the solvent content is 44.3% and the tetramers
have multiple contacts with their neighbors. Crystal
packing contacts are numerous across the entire tetra-
mer surface and include some weak contacts in the
loop region (residues 22–42). The crystal contacts in
form 1 leave sufficient room for unhindered packing of
the loops in the b-hairpin conformation.
In crystal form 2, the contacts are much more

sparse, despite having almost the same solvent content
(43.6%) as crystal form 1. Two important lattice
Fig. 2. Schematic representation of the
crystal packing in crystal form 2. Areas in
contact are enclosed in the boxes. Boxes in
broken lines represent symmetry-related
contacts to those enclosed by the solid
boxes. The contact on the left hand side
comprises the active site loop (residues
22–42), shown in detail in Figs 3 and 4. At
the other side of the tetramer, the active
site loops are not in direct lattice contact,
resulting in much higher temperature
factors. Both loops in the upper dimer,
regardless of the packing contact, are in a
helical conformation, whereas the lower
dimer loops are in an extended
conformation.
Fig. 3. The view of the active site of both superposed TM1415
models (crystals forms 1 and 2) and that of human IMPase (PDB
code 1AWB). The model of the human enzyme (in light gray) has
three metal ions bound (yellow spheres) and the substrate (
D-inosi-
tol-1-phosphate, gray). The model of the TM1415 crystal form 1 is
in purple and the crystal form 2 in green (including the tartrate
molecule). The conformational change in loop 22–42 is indicated by
green arrows, and the change in the 60s loop harboring Glu65–66
directly co-ordinating metal ions, is shown in red arrows. The con-
tact of the active site loop (22–42) in the helical (in green, crystal
form 2), as well as in the extended conformation (in purple, crystal

form 1), to the symmetry mate in extended conformation is marked
by a blue arrow.
Structure of TM1415 IMPase K. A. Stieglitz et al.
2464 FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS
contacts occur around t he loop region (residues 22–42).
The loops on one side of the tetramer are in direct and
intimate lattice contact (two direct hydrogen bonds),
whereas the loops on the other side of the tetramer are
practically unhindered, without direct contact (Fig. 2).
Even though the crystal packing in both forms is dif-
ferent, in form 2 there is still sufficient space around
the loops so that the lattice could accommodate the
tetramer with all loops in b-hairpin conformation,with-
out significant structural changes (Fig. 3).
Active site loops
The direct comparison of both crystal forms provides
unexpected insights into the principles of protein
design and shows an unusual structural plasticity. The
tetramer in crystal form 1 (P2
1
) is very symmetrical
(the rmsds between individual subunits are listed in
Table 2), whereas the tetramer in the crystal form 2 is
very asymmetrical. In the following discussion, we use
the nomenclature suggested in Fig. 4B, which identifies
the upper and lower dimers in the tetrameric structure.
In crystal form 2, the lower dimer has the active site
loops in the b-hairpin conformation, whereas in the
upper dimer the loops are folded into two short a-heli-
ces (Figs 3 and 4). The color brackets in Fig. 4

indicate the same fragment folded in different confor-
mations in lower and upper dimers.
Despite lacking any significant contacts, loops in the
upper and lower dimers of crystal form 2 have distinct
and different conformations (Fig. 4). The loop (resi-
dues 62–72) shows significantly changed architecture
from a more extended conformation (b-sheet in form
1) to the more coiled conformation in form 2. The act-
ive site loop (residues 22–42), and the loop (residues
62–72) in TM1415 are required for the formation of
the active site in IMPase and FBPase [8,10]. Figure 3
shows the superposition of both crystal forms of
TM1415 and the human IMPase with a full comple-
ment of metal ions and the substrate. The structures
superpose well at the active site region and the figure
shows the change in conformations of those two active
site loops (green and red arrows). Both loops harbor
the residues involved in metal ion binding and there-
fore the subunits with alternative loop conformation
would be incapable of binding the full set of metal
ions. Additionally, residue Asp44, which plays a cru-
cial role in the catalytic mechanism (creation of the
nucleophile) in both upper subunits, has an alternative
conformation in which it loses a direct contact to
Thr84 Oc that is critical for water activation. All of
these conformational changes make the upper dimer
catalytically incompetent.
The catalytic loops in crystal form 1 have tempera-
ture factors slightly higher than, but comparable with,
the rest of the structure (Table 1). The stability of the

loops is confirmed with the quality of the electron
A
B
Arg24B
α
β
Arg24B
Asp42B
Asp42B
Lys31B
Lys31B
Lys30C
Asp42C Asp42C
Arg24CArg24C
Lys30C
Fig. 4. (A) The stereo-diagram of the active site loop (residues 22–
42) in contact with the same sequence symmetry-related fragment
(residues 22–42) covered with the sigma-weighted 2Fo-Fc electron
density, contoured at the 1.1 r level. The helical (residues 22–42)
fragment (blue model) is covered with the purple map, whereas
the symmetry-related 22–42 fragment (purple model) is covered
with a yellow map. The blue and red brackets denote the same
sequence in both models. (B) Comparison of both crystal forms.
The crystal form 1 is in purple and the crystal form 2 is in green.
Please note significant displacement of residues 22–42 (loop 1) and
62–72 (loop 2) in the upper dimer. The fragments enclosed in
boxes (with colors corresponding to the colors of the electron den-
sity maps in (A) are in contact in the crystal form 2, as presented
above.
K. A. Stieglitz et al. Structure of TM1415 IMPase

FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS 2465
density (Fig. 4 and supplementary Fig. S1). The tem-
perature factors for the loops in direct contact in crys-
tal form 2 (one in b- the second in a helical
conformation) are slightly higher than, but comparable
to, the rest of the structure; the remaining two loops
have substantially higher temperature factors (supple-
mentary Fig. S4) and much weaker electron densities.
The symmetrical crystal form 1 has a single metal
ion bound at the active site. This metal contributes to
the stabilization of active site architecture. Choe et al.
[10] concluded that this particular metal ion-binding
site is the most frequently occupied and has the highest
affinity to metal ions in FBPase. The metal ion does
not make direct contact with either of the two mobile
loops. In crystal form 2, we found no metal ions, but
a single molecule of tartaric acid in the active site cav-
ity. This ligand does not interact with any elements of
the active site loop. Its location is somewhat reminis-
cent of the sugar moiety of the substrate (Fig. 3).
Therefore, it would be difficult to argue that the tar-
trate or the metal ion is the cause of the asymmetry.
The asymmetry must originate from two differently
organized dimers present in solution that are dynamic-
ally incorporated into the lattice during crystallization
(supplementary Fig. S5).
Evolutionary link
The active site loop sequence (residues 22–42) is parti-
ally similar to the human enzyme and partially similar
to the sequences of M. jannaschii and A. fulgidus

IMPases (Fig. 5). In Fig. 5 we present the alignment
of the TM1415 sequence with sequences resulting from
the BLAST searches and clustering of all 786 inde-
pendent sequences into 13 functional families. Every
sequence in Fig. 5 represents a single protein family
that has distinct functionality. Figure 5 shows a small
fragment of the alignment, including the mobile loop
sequences. Judging by the clustering of sequences,
TM1415 is closer to the human IMPase. The mobile
loop sequence fits the environment less than other
fragments (as measured by psqs, data not shown). The
results of servers ranking this fragment propensity
for order-disorder (disprot) [22] clearly suggest that
this fragment has a tendency to be disordered. There-
fore, this fragment has evolved to be mobile and less
structured.
The characteristic element for our sequence is that,
when aligned structurally with other members of the
family, the fragment has low sequence conservation
(Fig. 5). It has variable length as well as amino acid
content, suggesting that the fragment is rapidly evol-
ving. Even when crucial positions are preserved, differ-
ent residues fulfill the same structural role (Figs 4 and
5). The other fragment (160s loop), predicted to be
unstructured (supplementary Fig. S4), participates in
the assembly of the dimer and also of the tetramer. As
Table 2. Comparison of the rmsds between different subunits
within individual crystal forms (off-diagonal terms), as well as
between corresponding subunits of both crystal forms (diagonal
terms). Please note the symmetry of crystal form 1 and asymmetry

of crystal form 2 represented by low and high values of the rmsd,
respectively.
Subunit
a
Crystal form 2 (P2
1
2
1
2
1
)
ABCD
Crystal form 1 (P2
1
) A 0.931 0.739 3.937 3.335
B 0.368 0.498 3.705 3.133
C 0.274 0.370 3.175 2.253
D 0.402 0.381 0.534 2.841
a
The rmsds were calculated on the 252 Ca positions of individual
subunits.
Table 1. Data collection and refinement summary.
Crystal form
12
Space group P2
1
P2
1
2
1

2
1
dmin (A
˚
) 2.2 2.4
Reflections unique 46 203 36 088
<I ⁄ r(I)>
a
14.1 (2.1) 10.1 (1.1)
Completeness (%)
a
98.3 (95.5) 98.0 (96.5)
Average redundancy
a
3.5 (2.2) 2.5 (2.3)
Unit cell (A
˚
) a ¼ 62.02 a ¼ 83.81
b ¼ 103.87 b ¼ 97.94
c ¼ 80.51 c ¼ 122.16
(deg) b ¼ 101.83
R
merge
0.058 (0.41) 0.063 (0.55)
R-factor 0.209 0.178
Free R-factor (5%) 0.279 0.266
rmsd
Bond lengths (A
˚
) 0.007 0.006

Bond angles (A
˚
) 1.37 1.25
Ramachandran plot (%)
Most preferred 89 89
Allowed 10 10
Generously allowed 1 1
Disallowed 0 0
Ligands and solvent
Ligands None 4 tartaric acid
Metal ions 4 None
Water molecules 303 299
Temperature factors (A
˚
)
Protein average 45.3 40.5
Ligands ⁄ metals 33.2 52.4
Loops 55.0 65 ⁄ 85
2
a
Last shell data in parenthesis (2.2–2.27 A
˚
, crystal form 1; 2.4–
2.49 A
˚
, crystal form 2).
b
Loops in contact ⁄ loops not in contact.
Structure of TM1415 IMPase K. A. Stieglitz et al.
2466 FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS

presented above, the change in the aggregation state
contributed to a change in enzyme functionality. The
inhibition, by Li
+
, of several members of the family,
must also be considered as an acquisition of a novel
functionality. In summary, the low sequence conserva-
tion and high variability of both loops when linked to
their structural instability suggests evolution of novel
functionalities being driven by changes in sequence of
those fragments [15,16,23].
Experimental procedures
Cloning and protein production
The TM1415 protein was expressed in E. coli, as described
previously [6]. After cell lysis and removal of the cell
debris by centrifugation, the supernatant containing
TM1415 was heated to 85 °C and the denatured E. coli
proteins were removed by centrifugation. The enzyme was
further purified by column chromatography on a Sepharose
QFF column. The protein was > 96% pure, as measured
by SDS ⁄ PAGE.
Crystallography
The protein was crystallized by the standing drop vapor
diffusion method. Five microlitres of the enzyme, in 50 mm
Tris buffer, pH 7.5, was mixed with 5 lL of the crystalliza-
tion buffer containing 15% (w ⁄ v) PEG 4000, 15% (v ⁄ v)
PEG 400, 0.2 m MgSO
4
, and 50 mm (NH
4

)
2
SO
4
,in50mm
cacodylate buffer, pH 8. The crystals appeared in 1 week
and had the morphology of chunky prisms with a lattice
organized by P2
1
symmetry. Mixing 5 lL of the enzyme in
50 mm Tris, pH 7.5, with 5 lL of the crystallization buffer
containing 15% (v ⁄ v) PEG 400 0.2 m sodium tartrate, and
0.2 m (NH
4
)
2
SO
4
in 50 mm citrate, pH 5.6, resulted in crys-
tals with the morphology of thin plates characterized by the
P2
1
2
1
2
1
space group. The data were collected to 2.2 A
˚
reso-
lution for the first crystal form, and to 2.4 A

˚
for the second
form, on the RaxisIV++ area detector mounted on the
Rigaku rotating anode at the X-ray facility at Rice Univer-
sity (Houston, TX, USA). The structure was solved by
molecular replacement (molrep), using the model construc-
ted from the structures of several IMPases (human,
MJ0109 and AF2372) as search probes. The sequence of
TM1415 shares the highest similarity to human IMPase
( 26% identity) and therefore this molecule was used as a
scaffold. However, there are three deletions (residues 70s,
160s and 210s), making it a bit shorter. Those crucial dele-
tions are shared with MJ0109 and AF2372. Therefore, we
used those molecules to construct the fragments not corres-
ponding well to the human structure. The probe model was
built from fragments of those proteins with the highest
homology to TM1415. The full atom model was used for
molecular replacement and later refined, using cns,toa
standard R-factor of 0.209 and 0.178, as calculated on all
reflections to 2.2 and 2.4 A
˚
resolution, respectively, for
forms 1 and 2. The data collection and refinement statistics
can be found in Table 1.
Sequence versus structure analysis
The iterated BLAST search was used to establish the mem-
bers of the multifunctional IMPase family. The groupings
followed the CDD classification of the NCBI (http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd). The seq-
uence to 3D structure fit was estimated by the program

psqs, developed at the Burnham Institute by A. Godzik.
Predictions of the disorder were performed at the web ser-
ver of the disprot (Dunker) [21].
We also carried out a clustering experiment in
which we investigated the entire IMPase ⁄ FBPase family.
The PFAM clan ‘inositol polyphosphate 1 phosphatase
like superfamily’ includes three PFAM domain families
(a) Inositol_P(PF00459), (b) FBPase(PF00316) and (c)
FBPase_glpX(PF03320). The Inositol_P family has 65
sequences in its seed alignment, representing 586 proteins;
and FBPase and FBPase_glpX each has 14 and 6 seed
sequences, representing 233 and 101 proteins.
Fig. 5. Multisequence alignment of the loop fragments of all the family representatives, including T. maritima and Homo sapiens. Amino
acids implicated in the catalytic mechanism are on a blue background. The green and the light blue background mark conserved residues.
The secondary structure, as calculated by
DSSP [26] is marked under the sequences, with red for the a-helix and orange for the b-sheet. The
figure was prepared using the program,
VISSA [27].
K. A. Stieglitz et al. Structure of TM1415 IMPase
FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS 2467
All these 85 seed sequences were clustered into sub-
families at 25% sequence similarity and one representative
sequence was selected for each subfamily. Within each clus-
ter, all the sequences were similar to the representative at
25% similarity or more, but similarities between all the rep-
resentatives of different clusters were below 25%. The clus-
tering was performed with program cd-hit [24].
These 85 sequences were clustered into 13 subfamilies.
Three families represented singletons, and another 10 sub-
families contained 2–22 members. Five subfamilies con-

tained proteins with 3D structures deposited in PDB. The
average identity within each cluster was 35%. Figure 4
shows the alignment of TM1415 (which belongs to the
IMPA1_HUMAN cluster) with representative proteins
from each cluster. The initial alignment was made using
clustalw [25] and was edited according to the structural
alignment of five present in the PDB proteins.
Acknowledgements
We would like to thank Dr Andrey Bobkov for help
in protein production and gel filtration experiments.
This work was supported by the NIH grant
1R01 G64481 (to BS), National Science Foundation
grant MCB-9978250 (to MFR), and Department of
Energy Biosciences DE-FG02–91ER20025 (to MFR).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Sigma-weighted 2Fo-Fc electron density, con-
toured at the 1.1 r level covering the active site loop
(residues 22-42), the active site residues (65-66, 79-84)
and the ligands bound.
Fig. S2. Ribbon representation of a single monomer of
the TM1415 (left) and the pig FBPase (right).
Fig. S3. Temperature factor plots for both crystal
forms.
Fig. S4. Diagram representing the disorder-score, as
calculated by the program disprot (Dunker) [22] .
Fig. S5. The chromatogram obtained from gel filtra-
tion experiments carried out in citrate buffer (pH 5.6)
on a Superdex 200 column.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-

ponding author for the article.
K. A. Stieglitz et al. Structure of TM1415 IMPase
FEBS Journal 274 (2007) 2461–2469 ª 2007 The Authors Journal compilation ª 2007 FEBS 2469