Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+.

Abstract

The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear.
We determined the crystal structure, to 2.6 A resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own.
The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.

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BioMed Central
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BMC Structural Biology
Open Access
Research article
Dimerization of inositol monophosphatase Mycobacterium
tuberculosis SuhB is not constitutive, but induced by binding of the
activator Mg2+
Alistair K Brown†1, Guoyu Meng†1,4, Hemza Ghadbane†1, David J Scott2,
Lynn G Dover1, Jérôme Nigou3, Gurdyal S Besra*1 and Klaus Fütterer*1
Address: 1School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK, 2National Centre for Macromolecular
Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington, LE12 5RD, UK, 3Department of Molecular Mechanisms of
Mycobacterial Infections, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique Unité Mixte de
Recherche 5089, Toulouse, France and 4Present address : School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK
Email: Alistair K Brown - a.k.brown.1@bham.ac.uk; Guoyu Meng - ubcg54l@mail.cryst.bbk.ac.uk; Hemza Ghadbane - HXG597@bham.ac.uk;
David J Scott - dj.scott@nottingham.ac.uk; Lynn G Dover - L.G.Dover@bham.ac.uk; Jérôme Nigou - jerome.nigou@ipbs.fr;
Gurdyal S Besra* - G.Besra@bham.ac.uk; Klaus Fütterer* - K.Futterer@bham.ac.uk
* Corresponding authors †Equal contributors
Abstract
Background: The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl
inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host
interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated
through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase).
Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function
of four IMPase-like genes in M. tuberculosis is unclear.
Results: We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis
SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the
paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence
of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the
crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively
promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce
dimerization on its own.
Conclusion: The correlation of Mg2+-driven enzymatic activity with dimerization suggests that
catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit
that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop
at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote
dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer
equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB
activity.
Published: 28 August 2007
BMC Structural Biology 2007, 7:55doi:10.1186/1472-6807-7-55
Received: 22 June 2007
Accepted: 28 August 2007
This article is available from: http://www.biomedcentral.com/1472-6807/7/55
© 2007 Brown et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
The enzyme inositol monophosphatase (myo-inositol-1-
phosphate phosphohydrolase, EC 3.1.3.25, IMPase) has
attracted considerable scrutiny since the early 1980s.
Then, it was discovered that low millimolar concentra-
tions of Li+ inhibit IMPase-catalysed dephosphorylation
of myo-inositol-1-phosphate [1], which could plausibly
explain the marked decrease of myo-inositol in brain tis-
sue following administration of lithium, a veteran thera-
peutic in manic depression treatment [2]. Inhibition of
myo-inositol synthesis affects the phosphatidyl inositol
second messenger pathway, which is linked to manic
depression, and it is now widely accepted, though
unproven, that IMPases constitute a major target of lith-
ium therapy [3].
The mycobacterial cell wall contains several lipid constit-
uents based on the structure of phosphatidylinositol (PI),
such as free PI, phosphatidylinositol-mannosides,
lipomannan, and lipoarabinomannan [4]. In addition to
their critical structural role, these lipids are significant as
immunomodulatory factors in interactions of the tubercle
bacillus with the host [5-7]. PI, an essential structural
component, is synthesised by phosphatidylinositol syn-
thase, M. tuberculosis PgsA, from CDP-diacylglycerol and
myo-inositol [8]. In mycobacteria, the supply of myo-inosi-
tol to PI synthesis is thought to be maintained by de novo
synthesis, which entails conversion of glucose-6-phos-
phate to inositol-1-phosphate, catalysed by inositol-1-
phosphate synthase, and subsequent dephosphorylation
of inositol-1-phosphate catalysed by an IMPase [9]. Thus,
IMPase activity is a critical part of PI biosynthesis in myco-
bacteria. Indeed, growth of Mycobacterium smegmatis is
accompanied by IMPase activity that dies off as growth
reaches the stationary phase, while growth is retarded in
the presence of lithium [10].
The M. tuberculosis genome encodes four IMPase-like
genes (Figure 1) [11,12], but little is known about the
function of the corresponding proteins in terms of M.
tuberculosis biology. The enzyme most closely related to
human IMPase (25% identity), M. tuberculosis SuhB
(Rv2701c), has been annotated as a 'putative extragenic
suppressor protein' according to its homology to E. coli
SuhB. The E. coli enzyme has been implicated in posttran-
scriptional control of gene expression [13], but no such
function has to date been described for M. tuberculosis
SuhB. According to the transposon mutagenesis study by
Sassetti et al., Rv3137 is essential, SuhB is dispensable
[14], and information on essentiality is as yet unavailable
for CysQ and ImpA. In vitro analyses showed that M. tuber-
culosis CysQ hydrolyses inositol-1-phosphate, adenosine-
monophosphate and fructose-1,6-bisphosphate [15]. In
contrast, SuhB appears to be a bona fide IMPase with little
or no activity towards fructose-1,6-bisphosphate. Still,
SuhB also de-phosphorylates a series of polyol phos-
phates including glucitol-6-phosphate, glycerol-2-phos-
phate, and 2'-AMP, albeit with significantly reduced
efficacy [12]. Like the human orthologue, SuhB is acti-
vated by Mg2+, and inhibited by lithium. Nevertheless,
SuhB requires higher concentrations of Mg2+ for full acti-
vation (at ~6 mM), and activity persists over a much wider
concentration range (~100 mM) of the activating ion
before it becomes inhibitory [12]. In order to better
understand potential functional differences between the
mycobacterial and eukaryotic IMPases, we have deter-
mined the crystal structure of M. tuberculosis SuhB and
characterised its self-assembly state in solution.
Results
Structure determination of SuhB
The structure of M. tuberculosis SuhB was determined by
molecular replacement to a resolution of 2.6 Å (Table 1,
Figure 2). The asymmetric unit of the crystal lattice con-
tains three independent copies of the SuhB monomer
forming two crystallographically distinct dimers: chains A
and C associate as one dimer, whereas chain B forms an
analogous dimer with one of its symmetry mates. The
quality of the refined model is limited by several factors:
first, a high Wilson B-factor (77.1 Å2) of the measured
structure factor amplitudes is mirrored by a high average
atomic displacement factor (78 Å2) (Table 1); second,
three disordered loop regions (see below) and mild disor-
der at either terminus leave 42 of 290 residues of the pri-
mary sequence unaccounted for (Figure 1); third, despite
a nominal resolution of 2.6 Å and reasonably strong data
in the high-resolution shell (Table 1), structural details are
less well defined by the (calculated) density than expected
at this resolution. In order to counteract an unfavourable
observables-to-parameter ratio, non-crystallographic sym-
metry (NCS) restraints were employed, leading to root
mean square (RMS) deviations between NCS-related mol-
ecules of 0.05 Å for backbone and ≤ 0.42 Å for side chain
atoms. Based on visual impression and real space R-factor,
the electron density is best defined for subunits A and C,
and worst for subunit B.
Apart from minor discrepancies, the SuhB monomer is
identical in fold to eukaryotic IMPases [16,17], with RMS
deviations between Cα positions of superimposed back-
bone structures ranging from 1.09 Å to 1.15 Å (Table 2).
In order to more readily compare SuhB with related struc-
tures we denote secondary structure according to the
recent report of the 1.4 Å-resolution structure of bovine
IMPase (Figures 1 and 2A) [17]. The IMPase fold is char-
acterised by alternating layers of α-helices and β-sheets in
an α-β-α-β-α sandwich arrangement (Figure 2A). Com-
parative studies established that IMPases share a structural
scaffold with inositol polyphosphate-1-phosphatases
(IPPases), fructose-1,6-biphosphatases (FBPases), 3'-

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phosphoadenosine-5'-phosphatases (PAPases) and 3'-
phosphoadenosine-5'-phosphatase/inositol-1,4-
bisphophatases (PIPases) (Table 2), although on the
amino acid sequence level the evolutionary relationship
between these groups of enzymes is not obvious [18-20].
However, members of this super-family differ notably
with respect to their assembly state: eukaryotic IPPases,
PAPases and PIPases are monomeric [19,21,22], eukaryo-
tic IMPases form homodimers as do a subset of dual activ-
ity IMPase/FBPases [16,17,23-25], whereas FBPases and
Sequence comparison of M. tuberculosis IMPase-like proteins with human IMPase
Figure 1
Sequence comparison of M. tuberculosis IMPase-like proteins with human IMPase. Sequences were aligned based
on the structural superimposition of SuhB and human IMPase using STRAP [53], and formatted using ESPript [54]. Secondary
structure elements of SuhB are above the sequence, cylinders representing helices and arrows β-strands, coloured according
to Fig. 2A. Full asterisks denote residues coordinating the catalytic Mg2+ sites, triangles indicate residues contacting inositol-1-
phosphate in the simulated SuhB-substrate complex. Horizontal boxes indicate disordered parts of the sequence. The grey
arrows indicate the positions of strands β1, β2 in the α1-α2 loop ('mobile loop') of human IMPase, with the open asterisk
marking the critical lysine residue.

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the dual activity IMPase/FBPase of Thermotoga maritima
form tetrameric assemblies [26,27].
Eukaryotic adenosine-monophosphate (AMP)-regulated
FBPases have been described in terms of two sub-
domains. In this description, layers I and II of the α-β-α-
β-α sandwich are equivalent to the AMP-binding sub-
domain of porcine FBPase, and layers III to V correspond
to the fructose-1,6-bisphosphate binding sub-domain
(Figure 2A) [26]. The primary sequence runs sequentially
through layers I and II of the 'AMP-binding' sub-domain,
but meanders in the fructose-1,6-bisphosphate-binding
sub-domain between layers III-V, with helices and strands
alternating. This pattern is interrupted only for strands
β12 and β13, which are not separated by a helix (Figures
1, 2A). The dimer observed in the crystal structure of SuhB
is primarily mediated by helices α6 in layer V and the β-
sheet in layer IV, which extends across the interface (Fig-
ure 2B). In contrast, strands in the sheet of layer II are ori-
ented perpendicular to the plane of the dimer interface.
Due to the absence of divalent metal ions and of substrate
in the active site, the loop connecting helices α1 and α2 is
disordered (residues 35–53 missing), as is much of the
loop connecting strands β3 and β4 (residues 85–96 miss-
ing). Both features are consistent with the structure of the
apo form of human IMPase (pdb entry 1IMF, [28]). Soaks
of SuhB crystals in Mg2+- or Mn2+-containing cryoprotec-
tion buffers failed to induce ordering, or give rise to den-
sity peaks associated with metals sites known from the
eukaryotic orthologues, which we attribute to the acidic
pH (4.1–4.5) of the crystallisation buffer. Further disorder
was seen in the hairpin loop connecting strands β12 and
β13 (residues 254–259) in layer IV, while density for the
β9-α6 loop was disjointed, yet indicating that the confor-
mation of the loop between residues 183 and 186 differed
markedly between subunits. As a consequence, side
chains for residues Tyr183, Val185, Arg186 and Cys187 in
this loop could not be built.
Active Site
The active site of SuhB is situated in a cavity carved out at
the N-terminal end of helix α7, sandwiched between lay-
ers II and IV (Figure 2A). Due to the disordered α1-α2
loop – referred to as the 'mobile loop' [27] – the active site
in SuhB appears wide open to solvent. Yet, it is anticipated
that the α1-α2 loop will become ordered upon the
enzyme binding metal ions and substrate. In this state the
active site is anticipated to be effectively shielded from
solvent, as is the case in the ligand-bound structure of
human IMPase [29]. In SuhB, the α1-α2 loop has a 10-res-
idue insertion relative to human IMPase (Figure 1).
Hence, the exact conformation of this loop in the ordered
state may well deviate from that of the human enzyme.
Interestingly, alternative secondary structure conforma-
tions of this loop were observed within the tetramer of the
structure of Thermotoga maritima IMPase/FBPase TM1415.
While the mobile loop included a short helix in two sub-
units of the tetramer, it assumed a β-hairpin conforma-
tion in the other two subunits [27]. The mobile loop
includes a lysine residue at position 36 (corresponding to
residue 49 in SuhB, marked by I in Figure 1) that stabilizes
the third of three catalytic metal sites through hydrogen
bonds to two ordered water molecules. In SuhB, this
lysine is conserved and parallels in the characteristics of
Li+ inhibition between SuhB and human IMPase (see Dis-
cussion) suggest an equivalent pattern of interactions in
the mycobacterial enzyme.
Structural elements of the active site required for metal
and substrate binding include the IMPase signature motif
104DPXDGT109 (superscripts denote residue numbers of
the SuhB sequence) between strand β4 and helix α4, the
82GEEG85 motif at the 'tip' of the β3-β4 loop, the 209G
[ST]AA212 motif in the β10-α7 loop, in addition to Glu228
in β11 and the 234WDXA237 motif in helix α8. All elements
Table 1: Crystallographic statistics
Beamline
Wavelength (Å)
Space group
Unit cell parameters
a, b, c (Å)
Resolution range (Å)
Unique reflections
Completeness (%)
< I/σ(I) >
Multiplicity
Rsymb (%)
Rcryst/Rfreec (%)
No. of non-hydrogen atoms
Protein
Solvent
Average B-factors (Å2)
Main chain – subunit A/B/C
Side-chain – subunit A/B/C
Solvent
Wilson B-factor
RMSD bonds (Å)
RMSD angles (°)
Ramachandran statisticsd
Core
Allowed
Generous
Disallowed
ID14-2 (ESRF, Grenoble)
0.933
C2221
101.5, 185.4, 106.9
20–2.60 (2.69–2.60)a
30931 (3028)
98.7 (97.4)
24.5 (2.5)
4.9 (3.8)
6.8 (25.8)
22.3/24.5
5466
5342
124
78
68/89/72
71/91/76
81
77.1
0.011
1.26
91.4%
7.9%
0.7%
None
a Numbers in parenthesis refer to the outer resolution bin.
b Rsym = ΣhΣi | I(h, i) - < I(h) >|/ΣhΣi I(h, i), where I(h, i) is the intensity
of the ith measurement of reflection h and < I(h) > is the mean value
of I(h, i) for all i measurements.
c Rcryst = Σhkl||Fo| - |Fc||/Σ |Fo|, where Fo is the observed structure-
factor amplitude and Fc the calculated structure-factor amplitude.
Rfree is calculated based on 5% of reflections not used in the
refinement.
d The Ramachandran plot was calculated using PROCHECK [43]

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contributing to metal site coordination are contained
within one subunit of the dimeric assembly. Compelling
evidence for three sites occupied by Mg2+ was provided
only recently by the 1.4 Å-structure of bovine IMPase [17].
This study, in conjunction with preceding structural stud-
ies on lithium-sensitive PAPase and lithium-insensitive
IMPase/FBPases [21,24,25,27], provided strong support
for a 3-metal catalytic reaction mechanism. When
mapped onto the structure of SuhB, these three magne-
sium ion sites are coordinated by Glu83 (equivalent to
residue 70 in human/bovine IMPase: h_70), Asp104
(h_90), Asp107 (h_93), Asp235 (h_220), in addition to
the carbonyl of Ile106 (h_92). In a previous study we had
shown that mutations to these sites in SuhB reduced activ-
ity dramatically [12]. Likewise, when activity was tested in
the presence of various divalent cations, Mg2+ was the
most potent activator of SuhB. These data and the high
level of sequence conservation among active site residues
justify the attempt to construct a model of inositol-1-
phosphate in the active site of SuhB (Figure 3A) and to
analyse potential differences in substrate-enzyme con-
tacts.
Fold and crystal dimer of M. tuberculosis SuhB
Figure 2
Fold and crystal dimer of M. tuberculosis SuhB. (A) Stereo ribbon diagram of the SuhB monomer, with secondary struc-
ture elements coloured according to the penta-layered αβαβα-sandwich arrangement of IMPases (cf. Figure 1). (B) Dimer of
SuhB formed by subunits A (blue, yellow) and C (lightblue, green). Selected secondary structure elements are labelled for ease
of comparison with panel A. Grey spheres indicate boundaries of disordered loops, with corresponding residue numbers in
black type. A (modelled) molecule of inositol-1-phosphate in ball-and-stick representation indicates the location of the active
site(s) in both panels.

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In our model of the SuhB-substrate complex, the phos-
phate moiety is positioned at the N-terminal end of helix
α4, the helix dipole countering the charge of the phos-
phate (Figure 3A). Most contacts between enzyme and
phosphate are through the three Mg2+ ions, with contact
distances in the order of 2.15–2.3 Å, in agreement with
the experimental structures of the eukaryotic IMPases
[17,28,29]. In addition, the phosphate forms H-bond
interactions with the amide nitrogen of Gly108 (h_94)
and Thr109 (h_95) at the N-terminus of helix α4. The
inositol moiety packs primarily against residues in the β9-
α6 and β10-α7 loops with specificity-determining con-
tacts provided by Glu228 (h_213) in strand β11, and
Asp107 (h_93) in the β4-α4 loop (Figure 3A). All con-
served side chains forming the metal binding sites or con-
tacting inositol-1-phosphate are seen in essentially the
same conformation as in the human/bovine enzyme, with
the exception of Glu83: as a consequence of disorder in a
large part of the β3-β4 loop, Glu83 (h_70) in subunit A
points outward rather into the active site (Figure 3A).
Significant discrepancies between the M. tuberculosis and
the human enzyme are seen in the β9-α6 loop, which in
human IMPase provides a contact surface, but no specifi-
city-determining interactions with the inositol ring [29]
(Figure 3B). The β9-α6 loop in SuhB is two residues
shorter than in the human enzyme with weak sequence
conservation (Figure 1). Although poorly ordered
between residues 183–186, residues in proximity to the
putative position of the inositol ring – Gly180, Phe181
and Gly182 – are well defined by density (Figure 3B).
Interestingly, the carbonyl oxygen of Phe181 falls within
H-bonding distance range of the C3 (2.9 Å) and C4 (3.3
Å) hydroxyls of the inositol ring, suggesting that the β9-α6
of SuhB may form specificity-determining contacts to the
substrate that are not present in the human enzyme.
A surprising observation was a patch of unexplained den-
sity in the active site that overlaps significantly with the
modelled position of inositol-1-phosphate, while a sec-
ond density peak coincides with the metal site 1 (Figure
3C) (in the numbering of ref. [17]). The latter is situated
between the carbonyl oxygen of Ile106 and the carboxy-
late of Asp104 and corresponds to the high-affinity metal
site [17]. The 'metal site 1' peak was present in all three
active sites, whereas the density for the inositol-1-phos-
phate site was best defined in subunit C (Figure 3C). We
consider it likely that the observed density represents a
weakly-bound molecule of octyl β-D-glucopyranoside, a
reagent used as an additive during crystallisation. Whether
or not the density peak a metal site 1 represents a divalent
cation could not be verified.
Dimer interface and assembly state
The present crystal structure comprises two apparent dim-
ers of SuhB. One dimer is formed between the NCS-
related subunits A and C, while the second dimer results
from crystallographic symmetry, pairing two symmetry-
related copies of subunit B. As a result of NCS restraints
applied during the refinement no substantial difference
between the two independent dimers is observed at the
interface. The SuhB dimers also superimpose closely with
the dimers of the human and bovine IMPase enzymes,
whereby the dimer interface is formed by analogous sec-
ondary structure elements. These include helix α6, the
strand β10, the β9-α 6 loop, helix α4, and the β10-α7 and
α7-β11 loops. In the superposition, which is based on sec-
ondary structure matching, helices α6 of the two protom-
ers are positioned opposite to each other (Figures 2B, 4A),
contributing approximately 30% of the buried surface
area (per monomer). It seems noteworthy that in SuhB
the axes of these two helices are spaced 7.5 Å apart
whereas in the eukaryotic enzymes that spacing is approx-
imately 10 Å (Figure 4A). The discrepancy can be attrib-
uted to alanine residues 192 and 196 being positioned at
the centre of this interface (Figure 4B), while the side
chains of a leucine at position 176 occupy the correspond-
ing space in human IMPase.
The interface of the SuhB dimer buries a total of 2696 Å2
of solvent accessible surface, calculated using the PISA
interface server [30,31], or 1348 Å2 per monomer. In
terms of buried surface area hydrophobic interactions
clearly dominate over H-bonds (12 contacts). Although
the residues located at the interface include a number of
polar and charged side chains (Figure 4C) only one ionic
Table 2: Comparison of M. tuberculosis SuhB to selected members of the Mg2+-dependent/Li+-inhibited phosphomonoesterase family of
proteins
IMPasec
FBPase IMPase/FBPasePAPaseIPPasePiPase
PDB IDa
1IMA2CZH 2BJI1FBP1DCU1LBV 1DK41QGX1INP1JP4
RMSD (Å) 1.09 (197)b
1.12 (199)1.15 (193)1.61 (159)1.88 (138)1.23 (177)1.13 (158)1.28 (190)1.41 (166)1.40 (182)
aReferences for the pdb entries cited in this table are provided in the supplementary information [see Additional file 3].
bNumber of aligned Cα positions.
cIMPase (inositol-1-monophosphatase), FBP (fructose-1,6-bisphosphatase), PAPase (3'-phosphoadenosine-5'-phosphatase), IPPase (inositol-
polyphosphate-1-phosphatase), PIPase (inositol-polyphosphate-1-phosphatase and 3'-phosphoadenosine-5'-phosphate phosphatase)

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interaction is seen. In human IMPase distinctly more sol-
vent accessible surface is buried in the interface (1675 Å2
per monomer). Contacts across the interface include 11
ionic interactions, while the number of H-bonds (13) is
about the same as in SuhB. One factor explaining the dif-
ference in size of the interface, at least in part, is the disor-
dered 'mobile loop' in SuhB. In the apo structure of
human IMPase (1IMF – [29]), the α1-α2 loop is also dis-
ordered, reducing the total of buried solvent accessible
surface per monomer from 1675 Å2 to 1571 Å2. Thus dis-
order of this loop accounts partially for the observed dis-
crepancy.
The analysis of the dimer interface using the PISA server
suggested that dimerisation of SuhB might not be consti-
tutive. Based on molecular contacts at the interface, PISA
Stereo views of the active site
Figure 3
Stereo views of the active site. (A) Active site of subunit A of SuhB, with metal sites (beige) and inositol-1-phosphate (col-
oured by atom type, carbons in light grey) modelled based on superimposition with the structures of bovine (PDB:2BJI, [17])
and human IMPase (PDB:1IMA, [29]). Residues in contact with metal sites and substrate in the modelled complex are shown as
sticks and numbered according to the SuhB sequence. Secondary structure elements are coloured as in Figure 2A. The loop in
violet indicates the approximate position of the α1-α2 loop, based on the superposition with PDB:1IMA [29]. (B) Bias-free dif-
ference density map of the β9-α6 loop in SuhB, calculated after simulated annealing of the model with residues 178–188
deleted and contoured at 2σ. The density is superimposed with apo SuhB (blue sticks) and human IMPase in complex with
inositol-1-phosphate (light green, PDB:1IMA, [29]). Putative (grey) and experimental (red) contact distances with substrate are
indicated. (C) Active site of subunit C of SuhB with σA-weighted Fo-Fc map contoured at 3σ, showing unexplained density
around the putative substrate position and metal site 1.

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calculates a 'complexation significance score' (CSS) that
on a scale from 0 to 1 indicates the probability that a pack-
ing interface generated by crystal symmetry might repre-
sent a 'real' interface [31]. The CSS for the dimer interface
of SuhB is 0.1, compared to a score of 1.0 for the dimer
interface of human IMPase (with or without the α1-α2
loop in the ordered state), suggesting that dimerisation
was merely due to crystal packing. We analysed self-asso-
ciation of SuhB by analytical ultracentrifugation (AUC) in
sedimentation velocity mode. To our surprise, but consist-
ent with the prediction by PISA, we found that SuhB is
predominantly monomeric, with the peak at molecular
weight 30,508 Da closely matching the calculated mass of
the SuhB monomer (Figure 5). However, with increasing
protein concentration a second peak at molecular weight
of 54,160 Da appears, and this peak becomes more pro-
nounced, at the expense of the main peak, when 1 mM
Mg2+ is added to the buffer (Figure 5). We verified by SDS
gel electrophoresis that the preparation of SuhB used in
this experiment did not contain a contaminant that could
explain the peak at 54,160 Da (data not shown).
These data suggested that SuhB, at protein concentrations
in the order of 1 mg.ml-1, exists in a monomer-dimer
equilibrium, but that Mg2+, which is required for activity,
might shift self assembly to the dimer state. The underes-
timate in molecular weight of the dimer peak is due to the
fitting of a single frictional coefficient to two species that
have differing frictional ratios. In addition, the appear-
ance of two separate peaks in the molecular weight profile
with increasing concentration indicates that the off-rate
for dimerisation is slow compared with the time of sedi-
mentation. Values for the off-rate for this regime are there-
fore estimated to be 10-5 < koff < 10-3 [32]. Analysing the
sedimentation velocity of SuhB in the presence of increas-
ing concentrations of Mg2+, confirmed that in the absence
of Mg2+ the monomer form is strongly preferred (Figure
6A). Yet, as Mg2+ increases in concentration (at a protein
concentration of 1.0 mg.ml-1) two features are observed:
first, the dimer peak increases in height, at the expense of
the monomer peak; second, the monomer and dimer
peaks shift towards each other until, at 5 mM Mg2+, a
broad skewed distribution of the sedimentation coeffi-
cient is observed (Figure 6A), suggesting a gradual transi-
tion from slow to fast exchange between the two assembly
states due to an increase in the off-rate of dimerisation.
Increasing the concentration of Mg2+ further, the dimer
peak eventually becomes the dominant species in the c(S)
distribution (Figure 6A).
Next, we examined whether the inhibitor Li+ influenced
dimerization. At an enzyme concentration of 1.0 mg.ml-1
Analysis of dimer interface
Figure 4
Analysis of dimer interface. (A) Helices α6 after superimposition of the dimers of human IMPase (cyan) and SuhB (red) by
secondary structure matching. The spacing of the helix axes is indicated in units of Å and the black oval indicates the position
of the non-crystallographic 2-fold axis mapping the subunits onto each other. (B) The helix α6 interface in SuhB with side
chains contributing to the contact surface indicated as sticks in cyan (subunit A) and yellow (subunit C). (C) Contact surface
(magenta) of subunit A calculated and visualised using Swiss PDB Viewer [50]. The active site indicated by a molecule of inosi-
tol-1-phosphate (yellow sticks).

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and in the presence of 5 mM Mg2+, increasing concentra-
tions of Li+ amplify the effect of Mg2+-induced dimeriza-
tion, with the dimer peak becoming dominant over the
monomer peak (Figure 6B). However on its own Li+ pro-
motes dimerization only very weakly, if at all (Figure 6B).
Furthermore the c(S) distribution obtained for 15 mM
Mg2+ matches almost perfectly the one obtained in pres-
ence of 5 mM Mg2+ and 5 mM Li+. Calcium, which binds
to IMPase on identical sites as Mg2+, but does not activate,
strongly promotes dimerization, while EDTA reverses
Mg2+-induced dimerization [see Additional file 1].
Discussion
The present crystal structure of M. tuberculosis SuhB con-
firms a highly conserved structural scaffold of IMPases in
mycobacteria with respect to overall fold and active site
geometry, in spite of a low level of overall sequence iden-
tity (25%) relative to human IMPase. The structural simi-
larity correlates with similar biochemical characteristics –
activation by magnesium, inhibition by lithium, specifi-
city for inositol-1-phosphate and exclusion of fructose-
1,6-bisphosphate as a substrate. It has been noted previ-
ously that the Mg2+-dependence of IMPase activity of
SuhB resembles more closely that of the thermophilic spe-
cies Thermotoga maritima, Archeoglobus fulgidus and Meth-
anococcus jannaschii [12,25,33,34] in that activation is
maintained over a concentration range of Mg2+ in the
order of 100 mM, whereas in eukaryotic IMPases Mg2+
becomes inhibitory above 5 mM. Such comparison
ignores, however, that unlike these thermophilic enzymes
SuhB does not hydrolyze fructose-1,6-bisphosphate, and
remains sensitive to Li+ (IC50 0.9 mM, [12]). Given that
the active site of SuhB, in its 'non-mobile' part, displays
the same highly conserved framework of side chains coor-
dinating the three metal sites as the eukaryotic ortho-
logues, we postulate that differences in Mg2+-dependence
of activity between SuhB and eukaryotic IMPases must be
linked on the one hand to structural differences in the
mobile α1-α2 loop, and secondly to the apparent link
Dimerization of SuhB is induced by Mg2+, or Mg2+ and Li+, but not by Li+ alone
Figure 6
Dimerization of SuhB is induced by Mg2+, or Mg2+ and
Li+, but not by Li+ alone. Traces of the sedimentation
coefficient distribution recorded in sedimentation velocity
experiments of M. tuberculosis SuhB. SuhB was at 1.0 mg.ml-1
in 20 mM Tris-HCl pH 7.9, 50 mM NaCl, plus MgCl2 or LiCl
as indicated. Samples were centrifuged at 40,000 rpm at 4°C
for at least 12 hours. The peak at 1.6 S corresponds to a
molecular weight of 30,147 Da, the peak at 2.5 S corre-
sponds to 50,116 Da.
Analytical ultracentrifugation in sedimentation velocity mode demonstrates that SuhB is monomeric in the absence of Mg2+
Figure 5
Analytical ultracentrifugation in sedimentation
velocity mode demonstrates that SuhB is mono-
meric in the absence of Mg2+. Main panel: Traces of the
molecular weight distribution of M. tuberculosis SuhB in a sed-
imentation velocity experiment. Conditions analysed were
0.5 mg.ml-1 SuhB (solid triangle), 1.0 mg.ml-1 SuhB (open tri-
angle),1.0 mg.ml-1 SuhB + 1 mM Mg2+ (open circle). The main
peak is centred at 30,508 Da, the secondary peak at 54,160
Da. Inset: enlarged view of the region between 40,000 and
80,000 Da. Samples were centrifuged at 40,000 rpm, 4°C, for
a minimum of 12 hours, in 20 mM Tris-HCl pH 7.9, 50 mM
NaCl and MgCl2 as indicated.

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between Mg2+-driven dimerization and activation of
SuhB. In IMPases and the tetrameric IMPase/FBPases the
α1-α2 loop, when ordered, stabilizes the third of the three
catalytic metal sites through bridging water molecules
[17,25,29]. The α1-α2 loop contributes significantly to
the dimer interface. This is apparent when comparing the
buried solvent-accessible surface in the dimer interface of
human IMPase between the ordered and disordered state
of the mobile loop. Thus, we anticipate that ordering of
the α1-α2 loop in SuhB, which is 10 residues longer com-
pared to human IMPase, will expand the dimer interface
and help stabilize the dimer state. While SuhB is driven to
the dimer state at high protein concentrations in the
absence of metal ions, as was the case during crystalliza-
tion, it is evident that Mg2+ strongly promotes dimeriza-
tion at low concentrations. Lithium amplifies the Mg2+-
induced effect, yet Li+ alone does not noticeably shift the
monomer dimer equilibrium. These observations corre-
late with the characteristics of metal binding in the active
site, which is known to promote ordering of the α1-α2
loop. According to 7Li-NMR binding data, lithium occu-
pies a single site per monomer in IMPase [35], and various
indirect evidence points to Li+ displacing Mg2+ from either
site 2 or site 3 [17,21,36,37]. A lysine residue in the
mobile loop (Lys36 in human, open asterisk in Figure 1)
that coordinates the third metal site via hydrogen bonds
to bridging water molecules has been shown to critically
influence Li+-sensitivity [37], and this lysine is conserved
in SuhB (Lys49). Given that the α1-α2 loop is disordered,
the present structure leaves open the precise geometry of
this loop in the ordered state and whether Lys49 in SuhB
coordinates the third metal site. Yet, lithium sensitivity of
SuhB (IC50 0.9 mM, [12]), the debilitating effect on activ-
ity of the W234A mutation, and the loss of Li+ inhibition
through the L81A mutation [12] – a mutation that
removes a stabilising hydrophobic contact to the con-
served Trp234 in the active site – hint that metal binding
induces ordering of the α1-α2 loop in SuhB in a similar
fashion as in the eukaryotic orthologues.
Previously, size exclusion chromatography experiments
with 1 mM EDTA present in the buffer had indicated that
E. coli SuhB formed monomers and it had been inferred
that the E. coli enzyme was active in the monomeric form
[13]. The parallels between metal-dependent self-associa-
tion and enzymatic activation in M. tuberculosis SuhB
strongly suggest that dimerization is linked to loading of
the three metal sites in the active site, and that phos-
phatase activity occurs in the dimer state, although we are
not in a position to determine whether dimerization is
required for activity. If dimerization were required for
enzymatic activity of SuhB, this would at least in part
explain the wide range of Mg2+ concentrations over which
activity is maintained, as the transition to the dimer state
is not complete up to at least 15 mM Mg2+. Several caveats
go with this rationale. First, the comparison of the activity
and AUC data is complicated by the fact that metal bind-
ing is cooperative with substrate binding [38] and inosi-
tol-1-phosphate was not present in the AUC experiments.
Also, while dimerisation appears to correlate with loading
of the metal positions in the active site, we cannot rule out
the possibility that the dimer interface contains one or
more metal binding sites, which could drive dimerisation.
However, the present structure, in line with related
IMPases, and a series of metal-soaking experiments with
SuhB crystals provided no indication for such a site (data
not shown).
Crystal structures of eukaryotic IMPases, a refolding study
and AUC analysis of human IMPase 2, in the absence of
Mg2+, consistently indicate constitutive dimerization of
the eukaryotic orthologue [16,17,23,39]. It is not clear
what mechanistic purpose dimerization serves with
respect to the enzymatic properties of IMPases, and why
dimerization should be constitutive in eukarya, but not in
bacteria. Unlike the tetrameric FBPases, which are regu-
lated by an allosteric mechanism involving changes in the
relative orientation of the subunits in the tetramer (see
[40] and references therein), no regulatory mechanism for
IMPase has been reported that invokes dimerization of
the enzyme. Nevertheless, Mg2+ has been shown to mod-
erately enhance thermal stability of M. tuberculosis SuhB,
which correlates to some extent with phosphohydrolase
activity of SuhB peaking at about 80°C [12]. While this
latter property is mirrored by E. coli SuhB [13] and M.
tuberculosis CysQ [15] it has not been tested whether at
such high temperatures SuhB still discriminates between
substrates. Thus, while dimerization may increase thermal
stability, the functional role of Mg2+-induced dimeriza-
tion of SuhB in the physiological regime of the tubercle
bacillus is not clear.
SuhB – a template for the other M. tuberculosis IMPases?
The IMPase-like genes in M. tuberculosis display consider-
able sequence diversity relative to each other. In the mul-
tiple sequence alignment (Figure 1), SuhB and Rv3137
align with 30% sequence identity, while ImpA and CysQ
score significantly lower (= 23%) relative to any sequence
in Figure 1 (Table 3). Searching the PDB for homologues
of known structure, using BLAST, showed that SuhB still
represents the best template in terms of sequence identity,
although the differences to the next nearest homologue
are small (Table 3). Based on the search results, one could
speculate on the substrate specificities of the two unchar-
acterised gene products, Rv3137 and ImpA. The gene
product with the most divergent sequence, CysQ, has
been characterised as a dual IMPase/FBPase [15]. Consist-
ent with this finding, searching the PDB with the CysQ
sequence returns two dual activity enzymes, Rattus nor-
vegicus 3'-phosphoadenosine-5'-phosphate/inositol-1,4-

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bisphosphate phosphatase (RnPIP) [22], and T. maritima
IMPase/FBPase TM1415 [18,27] as the closest homo-
logues of known structure. Likewise, the search results
mirror the close relationship of SuhB to the eukaryotic
IMPases, which strongly prefer inositol-1-phosphate as
substrate. Interestingly, the dual activity IMPase/FBPase
TM1415 appears as the top hit for both ImpA and Rv3137
(Table 3).
Analysing the multiple sequence alignment that underlies
Table 3 [see Additional file 2] more closely, it is interesting
to note that enzymes that are specific for inositol-1-phos-
phate and exclude fructose-1,6-bisphosphate carry a
glutamic acid or glutamine at position 228 (SuhB
sequence; residue 213 in human IMPase), whereas Asp,
His, Ser or Thr are found in enzymes that hydrolyze both
substrates or are specific for fructose-1,6-bisphosphate. In
the inositol-1-phosphate-bound structure of human
IMPase [29], the corresponding residue, Glu213, forms
hydrogen bonds to the C3 and C4 hydroxyls of the inosi-
tol ring. In addition to the H-bond between Asp93
(Asp107 in SuhB) and the C6-hydroxyl, these H-bonds
are the only side chain-mediated, specificity-determining
contacts between the inositol moiety and the enzyme. In
the structural superimposition it is evident that an Asp
(and likewise Ser, Thr or His) at this position is unlikely
to confer selectivity as the distance between the terminal
group of the side chain and the sugar hydroxyls becomes
too large. Discrimination against fructose-1,6-bisphos-
phate is rooted primarily in steric clashes of the 6-phos-
phate the β9-α6 loop. As a result of deletions relative to
human IMPase, a much shorter β9-α6 loop in FBPases
and dual activity IMPase/FBPases provide the space to
accommodate the second phosphate group. Thus, dele-
tions in this region in addition to substitutions of Glu/Gln
at position 228 could be indicative of dual specificity.
Analysing the sequences of ImpA and Rv3137 in light of
these considerations suggests that at least Rv3137 is
restricted to inositol-1-phosphate, whereas the case is less
clear cut for ImpA. The latter carries a Ser at the position
corresponding to Glu228, but displays no significant dele-
tion relative to SuhB between residues 150 and 190,
whereas CysQ by the standards of both criteria falls into
the category of the dual specificity enzymes, consistent
with published biochemical data [15].
Conclusion
We have determined the structure of M. tuberculosis
SuhB, providing a structural template for the four IMPase-
like enzymes in this organism. While resembling eukary-
otic IMPases in terms of structural scaffold, specificity for
inositol-1-phosphate, requirement for Mg2+ and inhibi-
tion by Li+, SuhB clearly diverges from the paradigm of
constitutive dimerization of bona fide IMPases. The
present data support a model of Mg2+-dependent dimeri-
zation, where loading of the three catalytic metal sites in
the active site induces ordering of the mobile loop, pro-
moting dimerization likely by expanding the dimer inter-
Table 3: BLAST search for M. tuberculosis IMPase homologues of known structure
SuhBRv3137ImpACysQ1IMAa
2BJI 1JP4 2P3N1G0H
SuhB
Rv3137
ImpA
CysQ
1IMA (IMP human)
100 30
100
23
23
100
22
18
13
100
25
23
15
14
100
25
22
16
12
88
14
22
12
16
16
23
29
22
7
28
19
17
20
3
19
2.0E-20
(194)
3.0E-21
(194)
2BJI (IMP bovine)
4.7E-07
(165)
10019 2820
1JP4 (PIPase_Rno)
2.7E-05
(169)
8.7E-04
(192)
10018 15
2P3N(TM1415_Tma)
9.5E-16
(221)
4.0E-12
(247)
6.3E-10
(161)
10023
1G0H (MJ0109_Mja)
100
Fields above the table diagonal give the %-identity in a STRAP-generated multiple sequence alignment. Fields in bold type give the E-values and
(number of paired amino acid residues) of the two top hits in the BLAST search of the PDB [48].
a References for PDB entries:
1IMA – human IMPase complexed with myo-inositol-1-phosphate [29]
2BJI – bovine IMPase [17]
1JP4 – Rattus norvegicus 3'-phosphoadenosine 5'-phosphate and inositol-1,4-bisphosphate phosphatase [22]
2P3N – Thermotoga maritima TM1415 IMPase/FBPase [27]
1G0H – Methanococcus jannaschii MJ0109 IMPase/FBPase [24].

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face. The active site of SuhB presents a highly conserved
scaffold of side chains forming the metal- and substrate-
binding site, yet additional specificity-determining con-
tacts are expected with the weakly conserved β9-α6 loop.
Sequence and structural comparisons lead us to predict
that the essential gene product Rv3137 represents a bona
fide IMPase, while such may or may not be the case for
ImpA.
Methods
Materials
Centricon YM-10 filtration units were obtained from Mil-
lipore. E. coli C41(DE3) [41], acquired from Avidis
(France), was used for protein production in this study.
Sparse matrix crystallisation screens were obtained from
Molecular Dimensions Ltd. All other chemicals were rea-
gent grade or better and obtained from Sigma-Aldrich.
Expression, purification and crystallisation
Recombinant proteins were generated and purified as
described previously [12]. Purified His6-tagged SuhB, was
dialyzed extensively against 20 mM sodium phosphate
pH 7.5, 250 mM NaCl and purified further by size exclu-
sion chromatography over a Sephacryl S200-HR matrix.
Pure fractions were pooled and concentrated using Centri-
con YM-10 filter units. Crystals of SuhB were obtained by
hanging drop vapour diffusion, mixing 1 µl of protein
solution (40–50 mg.ml-1) with 1 µl of reservoir solution,
screening against commercial sparse matrix screens
(Structure Screen 1 and 2, Molecular Dimensions Ltd.) at
18°C. Initial crystals appeared in 30%(v/v)2-methyl-2, 4-
pentanediol(MPD), 0.1 M tri-sodium citrate, pH 5.6, 0.2
M ammonium acetate. Optimization led to crystals
approximately 0.3 mm in length at 10–14%(v/v) MPD,
0.1 M citric acid, pH 4.2, and 0.05%(w/v) octyl β-D-glu-
copyranoside. Crystals grew to their final size over 7–10
days. In preparation for X-ray data collection crystals were
soaked in cryoprotectant , consisting of upto 30%(v/v)
MPD, 125 mM NaCl, 0.1 M citric acid pH 4.2 and
0.05%(w/v) octyl β-D-glucopyranoside, increasing the
MPD concentration in steps of 10%. Crystals were
mounted in nylon loops and frozen in a 100 K nitrogen
gas stream.
X-ray data collection and crystallographic analysis
X-ray diffraction data for SuhB were recorded on beamline
ID14-EH2 at the European Synchrotron Radiation Facility
(ESRF), Grenoble, France (Table 1). Data were reduced
using DENZO/SCALEPACK [42]. Patterson self-rotation
analysis (POLARRFN – [43]) revealed a 3-fold non-crys-
tallographic rotation axis parallel to the 21-screw axis
(resulting in an apparent 6-fold), consistent with three
SuhB monomers per crystallographic asymmetric unit.
Structure factor amplitudes were normalized (ECALC –
[43]), and initial phases were obtained by molecular
replacement (AMORE – [43]), using monomer coordi-
nates of human IMPase (PDB:1IMA – [29]) as a search
model. While the Patterson cross-rotation search did not
reveal clear solutions, the correct 3 orientations (peaks 4,
5 and 7) could be identified through comparing rotation
angles between rotation function peaks with self-rotation
peaks, which was confirmed through the subsequent
translation search. The search model was stripped of non-
conserved side chains and insertions, and built manually
into MR-phased density (O – [44]). Search model bias was
minimised by building into simulated annealing omit
maps [45], followed by rounds of refinement (conjugate-
gradient minimization, simulated annealing) in CNS ver
1.1 [46] and manual rebuilding, eventually leading to a
model that fitted the data with an Rfree of ~30% (5% of
reflections). Non-crystallographic symmetry restraints
were introduced and the refinement was continued using
REFMAC5 [47]. The final model contains 3 monomers
covering SuhB residues 5–34, 54–85, 98–253, 260–286.
Details of the refinement statistics are listed in Table 1.
Coordinates and structure factors have been deposited in
the Protein Data Bank [48] [PDB:2Q74].
Molecular graphics
Figures 2, 3 and 4A,4B were prepared using PyMOL ver
0.97 [49]. Figure 4C was generated using Swiss PDB Viewer
[50] and POV-Ray Version 3.6 [51].
Analytical ultracentrifugation
Sedimentation velocity experiments were performed
using a Beckman Optima XL-A analytical ultracentrifuge
equipped with absorbance optics. Protein samples were
dialysed into storage buffer, as indicated in the figure leg-
ends (Figures 5 and 6) and loaded into cells with two
channel Epon centre pieces and quartz windows. Data
were recorded at 40,000 rpm, 4°C. A total of 100 absorb-
ance scans (280 nm) were recorded for each sample, rep-
resenting the full extent of sedimentation of the sample.
Data analysis was performed using the SEDFIT software
fitting a single friction coefficient [52].
Authors' contributions
A.K.B., G.S.B. and K.F. designed the study. A.K.B. wrote
the initial draft of the manuscript, and purified the pro-
tein. G.M. performed crystallisation experiments, data
acquisition and contributed to crystallographic analysis
and model building. A.K.B. and H.G. performed the AUC
experiment, and with help from D.J.S., analysed the data,
and prepared figures. L.G.D. together with J.N. designed
and produced the expression plasmids, developed the
purification protocol and contributed to data interpreta-
tion. K.F. solved the structure, contributed to design of fig-
ures, and edited the manuscript. All authors have read and
approved the manuscript.

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Additional material
Acknowledgements
We thank Tom Burton for his involvement in model building as part of an
undergraduate summer project. We thank Tim Dafforn for access to the
analytical ultracentrifuge, part of the Biosciences Biomolecular Characteri-
sation Facility (BBCF), and J. Baz Jackson for valuable discussions. We thank
ESRF Grenoble for access to the synchrotron and travel support, and ESRF
staff for support during data acquisition. G.M. was supported by an Adrian
Brown Scholarship. G.S.B. acknowledges support in the form of a Personal
Research Chair from Mr. James Bardrick, as a former Lister Institute-Jenner
Research Fellow, and the Medical Research Council.
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phosphatase activities: a novel target of lithium therapy. J
Mol Biol 2002, 315:677-685.
Arai R, Ito K, Ohnishi T, Ohba H, Akasaka R, Bessho Y, Hanawa-Suet-
sugu K, Yoshikawa T, Shirouzu M, Yokoyama S: Crystal structure
of human myo-inositol monophosphatase 2, the product of
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Additional file 1
Supplementary Figure S1. This Figure shows traces of the sedimentation
coefficient distribution recorded in sedimentation velocity experiments of
M. tuberculosis SuhB. SuhB was at 1.0 mg.ml-1 in 20 mM Tris-HCl pH
7.9, 50 mM NaCl, plus MgCl2, LiCl, CaCl2 and EDTA as indicated.
Samples were centrifuged at 40,000 rpm at 4°C for at least 12 hours.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1472-
6807-7-55-S1.png]
Additional file 2
Supplementary Figure S2. This Figure shows a structure-based sequence
alignment of IMPase-like proteins using STRAP [53] with formatting in
ESPRIPT [54]. Sequence abbreviations (with pdb accession code in
parentheses) are as follows: SuhB_Mtb – Mycobacterium tuberculosis
SuhB ; Rv3137_Mtb – gene product Rv3131 of M. tuberculosis;
ImpA_Mtb – M. tuberculosis ImpA; CysQ_Mtb – M. tuberculosis
CysQ; IMPase_hum – human inositol monophosphatase (1IMA);
IMPase2_hum – human inositol monophosphatase 2 (2CZH);
IMPase_bov – bovine inositol monophosphatase (2BJI); SuhB_Eco –
Escherichia coli SuhB; MJ0109_Mja – Methanococcus jannaschii
IMPase/FBPase MJ0109 (1DK4); AF2372_Afu – Archeoglobus fulg-
idus IMPase/FBPase AF2372 (1LBV); TM1415_Tma – Thermotoga
maritima IMPase TM1415 (2P3N); PIPase_Rno – Rattus norvegicus
3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate phos-
phatase (1JP4). Secondary structure elements above the sequence refer to
the crystal structure of M. tuberculosis SuhB.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1472-
6807-7-55-S2.png]
Additional file 3
References for Table 2. This file contains references for PDB entries cited
in Table 2 of the main text.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1472-
6807-7-55-S3.pdf]

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