doi:10.1016/S0022-2836(03)00729-0
J. Mol. Biol. (2003) 331, 385–393
Structure of Mycobacterium tuberculosis Singlestranded DNA-binding Protein. Variability in
Quaternary Structure and Its Implications
K. Saikrishnan1, J. Jeyakanthan1, J. Venkatesh2, N. Acharya2, K. Sekar3
U. Varshney2 and M. Vijayan1*
1
Molecular Biophysics Unit
Indian Institute of Science
Bangalore 560012, India
2
Department of Microbiology
and Cell Biology, and Indian
Institute of Science, Bangalore
560012, India
3
Bioinformatics Centre, Indian
Institute of Science, Bangalore
India 560 012
Single-stranded DNA-binding protein (SSB) is an essential protein
necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding
domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different
crystals distributed in two forms. The structure of one of the forms was
solved by a combination of isomorphous replacement and anomalous
scattering. This structure was used to determine the structure of the
other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the
molecule in different subunits in the two forms and those in Escherichia
coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar
structure, although the three loops exhibit considerable structural
variation. However, the tetrameric MtuSSB has an as yet unobserved
quaternary association. This quaternary structure with a unique dimeric
interface lends the oligomeric protein greater stability, which may be of
significance to the functioning of the protein under conditions of stress.
Also, as a result of the variation in the quaternary structure the path
adopted by the DNA to wrap around MtuSSB is expected to be different
from that of EcoSSB.
q 2003 Elsevier Ltd. All rights reserved
*Corresponding author
Keywords: single-stranded DNA-binding protein; homo-tetrameric SSB;
protein –DNA interactions; oligonucleotide binding fold; Mycobacterium
tuberculosis
Introduction
The DNA metabolising activities, like replication, repair and recombination, involve the
conversion of double-stranded DNA (dsDNA) to
single-stranded DNA (ssDNA). Single-stranded
DNA-binding protein (SSB) protects the transiently
formed ssDNA from nuclease and chemical
attacks, as well as prevents it from forming
aberrant secondary structures. The action of SSB is
of utmost importance in maintaining the genomic
integrity, which makes it one of the minimal gene
products that are required for life.1 The protein
has been identified in all classes of organisms performing similar functions but displaying little
Abbreviations used: SSB, single-stranded DNAbinding protein; dsDNA, double-stranded DNA.
E-mail address of the corresponding author:
[email protected]
sequence similarity and very different ssDNA
binding properties. Based on their oligomeric state
SSBs can been classified into four groupsmonomeric, homo-dimeric, hetero-trimeric and
homo-tetrameric.2 The bacterial and mitochondrial
SSBs constitute the homo-tetrameric class of SSBs.
A prominent feature of SSBs is the commonality in
the DNA-binding domain, which is made up of a
conserved motif, the OB (oligonucleotide binding)
fold.2 Adenovirus DNA-binding protein is the
only SSB with known structure that does not have
the OB fold.3
The crystal structure of the DNA-binding
domains of HMtSSB4 and EcoSSB5 – 7 indicate a
very high degree of structural conservation among
tetrameric SSBs, despite a low level of sequence
identity.6 The bacterial SSBs in general can be
divided into two domains, the N-terminal DNAbinding domain and the C-terminal glycine-rich
loop with an acidic tail. The C terminus is
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved
386
proposed to be involved in interaction with
enzymes of DNA metabolism.8 – 10 Mycobacterium
tuberculosis encodes a 164 residues long ssDNA
binding protein, MtuSSB. The protein exists as a
tetramer in solution with a molecular mass of
69 kDa. Limited biochemical studies indicate very
close functional similarities between MtuSSB and
the prototypical EcoSSB, in vitro.10,11 This is surprising given the low level of sequence identity
(, 28%) the two proteins share. Both the proteins
display similar DNA-binding affinities. However,
MtuSSB fails to complement the Dssb strain of
EcoSSB.9
M. tuberculosis,
the
causative
agent
of
tuberculosis, unlike many other pathogens, has
evaded detailed scrutiny at the molecular level,
primarily because of its long growth time,
fastidious growth requirement and handling risk.
It is only over the last couple of decades that
molecular genetic analysis of M. tuberculosis12 augmented by the availability of the complete
genome13 has revealed its unique metabolic pathways. The members of the M. tuberculosis complex
display very little genetic diversity, which has
been suggested to reflect a replication machinery
of high fidelity or a very efficient DNA repair
system.14 As part of a larger effort to understand
this unique and efficient phenomenon of DNA
metabolism, we have solved the three-dimensional
structure of MtuSSB in two crystal forms.
Structure of M. tuberculosis SSB
Figure 1. Ribbon diagram of the MtuSSB tetramer in
comparison with EcoSSB and HMtSSB. The three
dyad axes P, Q and R, and the four subunits are
labelled appropriately. The Figure was generated using
MOLSCRIPT27 and rendered using Raster3D.28
P, Q and R, as indicated in Figure 1. The tetramer
in form I is located on a crystallographic 2-fold
axis, which coincides with Q, such that subunits A
and B constitute the asymmetric unit. The asymmetric unit in form II contains two half-tetramers
(subunits A and C), as the two tetramers are
located on different crystallographic 2-fold axes,
Results and Discussion
Molecular architecture
The present analysis provides the structure of
MtuSSB in four different crystals distributed in
two forms. Two different sets of crystals in the
trigonal form (form I) were obtained from
solutions containing zinc and cadmium, respectively. Form II orthorhombic crystals could be
grown from either full-length protein or truncated
protein. The structure of crystal form I was solved
using multiple isomorphous replacement and
anomalous dispersion. Subsequently, a subunit
from form I was used to solve the structure of
form II by molecular replacement. Crystal form I
contains full-length protein, while form II contains
truncated protein with molecular mass ranging
from 14 kDa to 11 kDa.15 The C-terminal region
beyond amino acid residue 120, however, is undefined in both the structures. Even in the N-terminal
domain the entire length is not defined in any of
the subunits as a consequence of poor electron
density, which possibly is the cause for the comparatively higher R-factor (Table 2). A similar
situation obtains in the crystals of HMtSSB4 and
EcoSSB.5,7 However, all residues are defined in one
subunit or the other, providing a composite
structure of the N-terminal domain.
The MtuSSB tetramer is a dimer of dimers
having 222-symmetry with molecular 2-fold dyads
Figure 2. (a) Tertiary structure of MtuSSB with the
secondary structures and the three b-hairpin loops
labelled. (b) Structural superposition of the 12
crystallographically independent subunits of MtuSSB
from four different crystals depicting the extreme
mobility of the three loops. The superposition involved
the globular core of the protein.
Structure of M. tuberculosis SSB
387
Figure 3. (a) Structure-based sequence alignment of the three tetrameric SSBs, MtuSSB, EcoSSB and HMtSSB. The
secondary structure of MtuSSB is also depicted. EcoSSB is represented by two sequences as obtained from structures
solved independently by two different groups. See the text for details. Letters in small case indicate residues which
have not been defined in the crystal structure. (b) Structural superposition of the DNA-binding domain of the MtuSSB
in yellow, EcoSSB in magenta and HMtSSB in cyan. The superposition indicates a structurally conserved core with
flexible loops. The turn followed by a strand unique to MtuSSB directs the C terminus opposite to those found in
EcoSSB and HMtSSB.
coinciding with Q in one case and R in the other.
Thus, referring to Figure 1, subunits A and B
make up the asymmetric unit in form I, while subunits A and C from the two tetramers make up
the asymmetric unit in form II.
Topologically the N-terminal domain has the OB
fold (Figure 2(a)). Its structure is characterised by
three long b-hairpin loops extending out of a globular core formed by a five-stranded b-barrel, which
is capped by an a-helix. Loop 1 consists of residues
22– 27, loop 2 of residues 36– 52 and loop 3 of
residues 85– 98. The barrel is made up of strands
388
Structure of M. tuberculosis SSB
1, 3, 6, 7 and 8 with strands 1, 7 and 8 forming an
antiparallel b-sheet, which will be referred to as
the back b-sheet. The C terminus of the barrel
takes a turn and extends to a strand (strand 9)
forming a hook-like structure along with the preceding strand. The globular core has the same conformation in all the subunits (Figure 2(b)) with
r.m.s deviations (rmsd) ranging from 0.23 Å to
0.55 Å on pair-wise superposition. The three
loops, however, exhibit considerable variation in
their conformation and orientation with respect to
the core.
As the three-dimensional structure is better preserved through evolution than amino acid
sequence, a structure-based sequence alignment
gives a better result than that based on the amino
acid. Structural alignment of the MtuSSB with
different structures of EcoSSB and HMtSSB
(Figure 3(a)) revealed a difference between the
structures of EcoSSB reported by Raghunathan
et al.5,16 (PDB code 1KAW/1EYG), and Matsumoro
et al.7 (PDB code 1QVC). A gap at the position corresponding to residue 92 in MtuSSB was necessary
for good alignment in one case while it was not
required in the other. The number of sequence
identities from 92 to 110 in the first case was five
as against two in the second. Therefore, the structures reported by Raghunathan et al. were used in
Table 1. Surface area buried on oligomerisation (Å2).
(Values in the parentheses are hydrophobic buried
surface area)
comparative studies. The core of EcoSSB and
HMtSSB has structures similar to that of MtuSSB
with rmsd ranging from 0.77 Å to 1.29 Å. However,
the orientation of the C terminus of the DNA-binding domain with respect to the core, in addition to
its conformation, is distinctly different (Figure
3(b)). The polypeptide chain beyond strand 8 in
the two proteins is unstructured and is directed
opposite to that in MtuSSB.
Variability in quaternary association in SSBs
Though the tertiary structures are similar, the
quaternary structure of MtuSSB is remarkably
different from those of EcoSSB and HMtSSB
(Figure 1). Referring to Figure 1, the tetrameric
molecule with 222 symmetry can have three symmetrical non-equivalent interfaces: between A and
C (same as B and D); between A and B (C and D);
and between A and D (B and C). In a dimer of
dimers the subunits with the highest interfacial
area may be designated as a dimer. The remaining
two interfaces are those between the two designated dimers. In the case of HMtSSB and EcoSSB,
subunits A and C form the dimer.4,5 The surface
area buried at the three interfaces in MtuSSB is
given in Table 1. The surface areas buried when
the loops are omitted in the calculations are also
given in the Table. The values unambiguously indicate that subunits A and B constitute the dimer in
Pair-wise association
Structure
Tetramer
AC
AB
AD
8528
(5167)
6504
(4290)
6260
(3850)
1641
(1007)
812
(620)
1641
(1007)
2428
(1525)
2428
(1525)
1267
(862)
222
(56)
12 (0)
8568
(5155)
7295
(4782)
6091
(3724)
1691
(951)
1175
(808)
1691
(951)
2450
(1560)
2442
(1559)
1204
(864)
152
(49)
31 (24)
EcoSSB (PDB code
1eyg)
6490
(3539)
4768
(3079)
2333
(1185)
1472
(852)
417
(337)
417
(337)
659
(395)
659
(395)
HMtSSB (PDB code
3ull)
7853
(4240)
5538
(3162)
2777
(1482)
1620
(944)
588
(392)
588
(392)
836
(408)
836
(408)
MtuSSB (form I)
MtuSSB (form II)
222
(56)
152
(49)
The values obtained after removing the three b-hairpin loops
are given in the second line in all the cases, against each entry.
The third line in the case of MtuSSB contains values obtained
after removing residues beyond 110 (corresponding to 112 in
EcoSSB and 124 in HMtSSB). Undefined regions of the loops
were modelled in the case of MtuSSB. The residue ranges used
for these calculations are: MtuSSB, 3–118; EcoSSB, 1– 112; and
HMtSSB, 10–124. The values quoted for pair-wise association
are averages.
Figure 4. (a) Surface diagram of the MtuSSB dimer
illustrating the clamp-mechanism involving strand 9.
(b) Space-filling diagram of MtuSSB and EcoSSB
tetramers with the three loops removed, viewed down
axis P. Subunits BD are oriented identically in the two
structures.
Structure of M. tuberculosis SSB
389
Figure 5. Spatial disposition of
the three-stranded back b-sheet in
the tetramer and across the three
interfaces in different tetrameric
SSBs. Subunit A is coloured
magenta, B in cyan, C in green and
D in yellow. The gap between the
two sheets from subunits A and C
(B and D) are bridged by water
molecules (blue) in Mtussb form I.
both forms of MtuSSB, with or without the loops
being taken into consideration. The AC interface
contributes substantially in stabilising the tetramer,
while the contribution of the AD interface is negligible. Interfacial areas calculated after removal of
residues beyond 110 emphasise the role played by
this region towards dimerisation in MtuSSB.
A unique clamp-mechanism involving strand 9
further stabilises the dimer AB in MtuSSB. The
strand from one subunit anchors to a cleft formed
by the helix and strands 8 and 9 of the other subunit (Figure 4(a)).
Grossly, the EcoSSB tetramer can be obtained
from the MtuSSB tetramer by rotating together
subunits A and C by 428 about P while keeping
subunits B and D unaltered (Figure 4(b)). The variation in the quaternary structure of SSB is perhaps
best explained in terms of the dispositions of the
back b-sheet across the three interfaces and in the
tetramer in the different SSBs (Figure 5). In EcoSSB
and HMtSSB, dimerisation involves the side-byside arrangement of the two three-stranded back
b-sheets with the formation of a contiguous sixstranded b-sheet. One of the two dimer– dimer
interfaces has a clear back-to-back arrangement of
b-sheets. In MtuSSB, however, dimerisation
involves the back-to-back arrangement of the two
sheets, and the formation of the dimer of dimers
primarily involves a side-by-side arrangement of
the sheets. While the two back b-sheets come
together to make a contiguous six-membered
b-sheet at the AC interface in form II, there is a
gap between the neighbouring strands from the
two sheets in form I. This gap is filled by water
molecules, which form bridges between the two
strands (Figure 5). The significance of this difference is not immediately obvious, though it may be
the result of packing the acidic C-terminal tail,
which is truncated in form II.
The distinct quaternary association observed in
MtuSSB results in a greater buried hydrophobic
surface area on tetramerisation (Table 1). The
buried hydrophobic area at the interfaces of the
tetramer is 1628 – 927 Å2 more in form I MtuSSB
than in EcoSSB and HMtSSB, respectively. The corresponding values in form II MtuSSB are 1616 Å2
and 915 Å2. Assuming the interface stabilisation
free energy upon burial of hydrophobic surface to
be 2 15 cal/mol per Å2,17 the MtuSSB tetramer is
expected to be more stable than EcoSSB and
HMtSSB tetramers by 14 – 24 kcal/mol. Unfolding
studies of MtuSSB in the presence of varying concentrations of guanidinium hydrochloride indicate
the protein to be more stable than EcoSSB.18
The charge distribution at the tetrameric interface formed by the six-stranded back b-sheet in
MtuSSB is considerably different from that in the
other two SSBs. In particular, a pair of charged residues, Lys7 and Glu80, and Arg16 and Glu95,
occurring in EcoSSB and HMtSSB, respectively, is
polar (Thr6 and Ser80) in MtuSSB. By virtue of the
molecular 222 symmetry these residues form a
cluster of alternating positive and negative electrostatic charges at the dimer– dimer interface of
EcoSSB/HMtSSB, which get buried on tetramerisation. Salt-bridges formed by these residues across
the dimer –dimer interface lock the orientation of
the subunits.4,5 In a quaternary structure of the
type found in MtuSSB, the ionic side-chains fail to
form salt-bridges, thus, compromising the structural stability. Introduction of a free ionic residue
in this region has been demonstrated to destabilise
the EcoSSB tetramer and results in formation of
stable dimer.19
Clamping involving strand 9 fastens the orientation of the subunits of the dimer in MtuSSB.
Also, Arg76 and Glu105 at the edge of the dimeric
interface form inter-subunit salt-bridges that add
to the stability of the dimer. Sequence alignment
indicates that the two charged residues at positions
corresponding to 7 and 80 in EcoSSB, are present in
SSBs of Gram-negative bacteria, of higher eukaryotic mitochondria and, to a certain extent, of low
G þ C-rich Gram-positive bacteria, indicative of
the conservation of the quaternary structure in all
these organisms. The SSBs from high G þ C-rich
390
Gram-positives do not have charged residues at
these positions.
A model for DNA-binding
A model of MtuSSB –ssDNA complex was constructed based on the structure of EcoSSB bound
to two strands of ssDNA.16 In the structure of the
EcoSSB complex, a 28-mer ssDNA wraps around
subunits A and C and a 23-mer around subunits B
and D. The AC subunits with the 28-mer were
superposed on the AC and BD subunits of MtuSSB.
On account of the 2-fold symmetry that relates A to
C and B to D, and as the oligonucleotide is asymmetric, this can be done in two ways, leading to
two possible MtuSSB – ssDNA models. In addition,
the crystallographically determined EcoSSB –
ssDNA model was completed by building in missing segments on the basis of the proposals of
Raghunathan et al.16. All the three models were
then refined as indicated in Methods.
Due to the variation in quaternary structure, the
globular core of MtuSSB tetramer, which binds to
DNA, can be approximated to an ellipsoid, while
the core of EcoSSB tetramer can be approximated
to a sphere (Figure 6). Differences in the shape of
the DNA-binding surface affect the length of the
bound ssDNA and the path taken to wind on to
the tetramer. In the case of EcoSSB, 61 nucleotides
were required to wrap the tetramer, while only 56
and 55 nucleotides were required to wrap the two
models of MtuSSB, respectively. While EcoSSB
tetramer binds to 65 ^ 3 nucleotides at physiological conditions,20 there are no reports on the
number of nucleotides required to bind to a
tetramer of MtuSSB.
Trp40 and Trp54 play a critical role in stabilising
EcoSSB –ssDNA complex by involving in base
stacking.16 The equivalent residues in MtuSSB are
Ile39 and Phe54. Despite the absence of these
tryptophan residues, which is expected to decrease
the binding affinity,21 MtuSSB has a DNA-binding
affinity similar to EcoSSB.10,11 From the model of
MtuSSB – ssDNA it appears that the substitution of
Trp54 is compensated by the tryptophan at
Figure 6. The two possible models of MtuSSB –ssDNA
and that of EcoSSB – DNA complex are illustrated. Also
depicted are the approximate ellipsoids that encompass
the globular core of the proteins. The 50 end of the
ssDNA is anchored to the subunit in yellow. The Figures
were generated using Ribbons29 and rendered using
POVRAY.
Structure of M. tuberculosis SSB
position 60, which is stacked against a base. The
equivalent residue in EcoSSB is phenylalanine.
There are ten basic residues (arginine and lysine)
per subunit in proximity to ssDNA bound to
MtuSSB, as opposed to seven in EcoSSB –ssDNA
complex. The additional ionic interactions, arising
from the higher number of basic residues on the
DNA-binding surface, may normalise the loss in
affinity due to the absence of tryptophan in
MtuSSB at position equivalent to Trp40 of EcoSSB.
Biological significance of the unique
quaternary association of MtuSSB
M. tuberculosis, in addition to growing within
host cells such as macrophages, has the ability to
enter into a prolonged state of dormancy, which is
thought to be a crucial component of mycobacterial
virulence.12 During the period of growth as well as
dormancy the organism has to withstand considerable environmental stress. Also, the metabolic processes, including protein synthesis, are minimal
during dormancy. Due to the essentiality of SSB in
safeguarding the genomic integrity from environmental stress, the protein is required to be everpresent and active. Mutation studies in EcoSSB
indicate a direct correlation between stability of
the tetramer and efficient DNA-binding.18,22 The
unique quaternary association of MtuSSB lends a
greater innate stability to the protein and consequently an increased half-life. During dormancy,
when the amount of protein is expected to be low,
expression of highly stable SSB would benefit the
organism. The G þ C-rich genome of M. tuberculosis
also dictates the requirement for an efficient SSB,
as ssDNA with higher G þ C content tends to
form larger and more stable secondary structures.
Complementation studies have demonstrated
the incapability of MtuSSB to perform the function
of EcoSSB and vice versa.9 The maintenance of
species specificity is attributed to the C-terminal
region of the protein, though chimeras constructed
by swapping the C-terminal domain of MtuSSB
and EcoSSB failed to overcome the barrier.9 Based
on the variation in quaternary structure of EcoSSB
and MtuSSB, we propose that the N-terminal
domain, in addition to other factors, contributes to
the maintenance of the species barrier. The crystal
structures of MtuSSB, for the first time, reveal significant structural variations among tetrameric
SSBs. It is possible, in theory, to take advantage of
these variations to generate peptidomimics of the
DNA that bind selectively and irreversibly to
regions unique to MtuSSB and disable this
essential protein.
Methods
Structure determination and refinement
MtuSSB was crystallised in two different crystal forms
and diffraction data were collected and processed as
391
Structure of M. tuberculosis SSB
Table 2. Data statistics
Form I
Data set
Space group
a (Å)
b (Å)
c (Å)
Data resolution (Å)
Unique reflections
Completeness (%) (final shell)
Rmerge (final shell)
Multiplicity (final shell)
Rmeasureb (final shell)
Phasing powerc
Refinement
R-factor (Rfree)
Resolution range (Å)
RMS deviation from ideality
Bonds (Å)
Angles (deg.)
Form II
Zinc
Cadmium
Mercury
Aa
Ba
P3121
78.7
78.7
77.2
2.50
9456
99.1 (99.9)
8.1 (41.0)
4.8 (3.0)
8.9 (52.4)
P3121
79.5
79.5
78.4
2.60
9072
99.1 (96.5)
10.9 (53.8)
7.3 (7.0)
11.7 (57.9)
0.85
P3121
I212121
60.4
117.6
175.2
2.70
17,174
97.9 (97.6)
7.3 (58.2)
4.4 (4.4)
8.3 (66.4)
I212121
60.2
116.7
177.9
3.20
10,610
98.9 (96.9)
10.1 (46.1)
4.6 (3.6)
11.4 (53.7)
22.8 (28.8)
15.0–2.50
21.2 (27.5)
15.0–2.60
23.1 (29.5)
15.0–2.70
23.5 (31.3)
15.0–3.20
0.007
1.7
0.007
1.6
0.008
1.6
0.009
1.7
91.8
6.2
1.6
93.4
5.2
1.4
90.5
6.5
3.0
2–41; 46 –120
2–43; 47–87; 97 –123
3–42; 49–86; 97–124
3 –44; 46–87:;96–120
3–39; 51–90; 97 –125
3–36; 51–87; 97–123
3–37; 51 –121
2–36; 51–85; 91 –118
3–40; 51–91; 95 –121
3–38; 51–87; 97 –118
Percentage of residues in Ramachandran plotd
Allowed region
93.0
Generously allowed region
5.5
Disallowed region
1.5
Residues defined
Subunit A
3–41; 47–120
Subunit B
2–91; 95–123
Subunit C
2.90
6301
97.0 (100)
10.4 (45.1)
8.9 (9.6)
11.1 (46.2)
1.25
Subunit D
a
B grew from crystallisation experiments involving the full-length protein after several months, while A was obtained from experiments using truncated protein.
b
As defined.30
c
Resolution range 10–4.0 Å.
d
Calculated for non-glycine and non-proline residues using PROCHECK.31
described by Saikrishnan et al.15. The data collection
statistics are given in Table 2. A good single-site mercury
derivative of the trigonal crystals grown in the presence
of zinc sulphate was obtained.15 A crystal grown in the
presence of cadmium sulphate was used as the other
derivative. Anomalous data obtained from both the
derivatives were used to improve the reliability of the
phases obtained from isomorphous differences. Harker
sections of the isomorphous and anomalous difference
Patterson maps indicated that each derivative contains a
single heavy-atom position. But the two positions were
different, making the derivatives independent of each
other. The position of the heavy atom in each case was
identified manually and confirmed using the routine
RSPS in the CCP4 program suite.23 Refinement of
heavy-atom parameters and phase angle calculations
were performed using MLPHARE.23 The anomalous
data from the two derivatives helped in identifying the
correct space group among the enantiomorphs. The
phases thus obtained were improved by solvent flattening using DM.23
Model building using FRODO24 was alternated with
iterations of rigid body refinement, positional refinement
and simulated annealing followed by individual temperature factor refinement using CNS1.1.25 Non-crystallographic symmetry restraints were applied at the initial
stages of refinement. Water molecules were defined
based on peaks with height greater than 2.5s in Fo 2 Fc
maps and those with height 0.8s in 2Fo 2 Fc maps.
The structure of form II was solved using AMoRe.26
This was followed by structural refinement using a protocol similar to the one mentioned above. The refinement
statistics are given in Table 2. Electron density in form I
containing zinc, corresponding to strand 9, is given in
Figure 7.
Structural superposition and accessible surface area
LSQKAB in the CCP4 suite of programs was used to
carry out structural superposition and calculate rootmean-square deviation (rmsd). The accessible surface
area of a molecule was calculated using NACCESS†
with a probe radius of 1.4 Å2. The buried surface area
was taken to be the difference between the sums of
accessible surfaces of individual subunits and that of
the oligomer.
† http://wolf.bms.umist.ac.uk/naccess/
392
Structure of M. tuberculosis SSB
Figure 7. Stereoview of the
electron density corresponding to
strand 9 from subunits A and B, in
the 2Fo 2 Fc map computed using
data from form I containing zinc.
The contours are drawn at 1s. The
Figure
was
prepared
using
FRODO.24
Modelling MtuSSB– ssDNA complex
All the missing regions of the N-terminal domain (2 –
119) of form II MtuSSB were modelled. Subsequent to
the docking of ssDNA, the models were soaked in a 4 Å
shell of water, using INSIGHTIIq, after the hydrogen
atoms were generated. The models were subjected to
energy minimisation and simulated annealing using
CNS1.1.25 A dielectric constant of unity was used
throughout. A main-chain restraint of 10 kcal/mol was
applied to the protein molecule. In the first step the
models were subjected to conjugate gradient energy
minimisation with a small, repulsive van der Waals
term introduced and the electrostatic term switched off.
In the next step, the electrostatic term was switched on
and the structures minimised for 100 cycles each. Subsequently, simulated annealing protocol was used to
remove ambiguities about the preferences of side-chain
and main-chain torsions among the available rotamers.
The models were heated to 3000 K and the simulations
were performed in steps of 25 K with each step containing 50 cycles spanning 5 fs each. Following simulated
annealing, one more step of conjugate gradient minimisation was carried out until the gradient of the total
energy was below 0.05 kcal/mol per Å.
Acknowledgements
The intensity data were collected at the X-ray
Facility for Structural Biology at the institute,
supported by the Department of Science and
Technology (DST) and the Department of Biotechnology (DBT). Facilities at the Supercomputer
Education and Research Centre, and the Interactive
Graphics Facility and Distributed Information
Centre (both supported by DBT) were used. This
work forms part of a genomics project sponsored
by the DBT.
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Edited by R. Huber
(Received 29 January 2003; received in revised form 23 May 2003; accepted 3 June 2003)