Structure of Mycobacterium tuberculosis Single-stranded DNA-binding Protein. Variability in Quaternary Structure and Its Implications

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 ˚ to r.m.s deviations (rmsd) ranging from 0.23 A ˚ on pair-wise superposition. The three 0.55 A 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 ˚ 2). Table 1. Surface area buried on oligomerisation (A (Values in the parentheses are hydrophobic buried surface area)

comparative studies. The core of EcoSSB and HMtSSB has structures similar to that of MtuSSB ˚ to 1.29 A ˚ . However, with rmsd ranging from 0.77 A 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

˚ 2 more in form I MtuSSB tetramer is 1628 – 927 A than in EcoSSB and HMtSSB, respectively. The cor˚2 responding values in form II MtuSSB are 1616 A ˚ 2. Assuming the interface stabilisation and 915 A free energy upon burial of hydrophobic surface to ˚ 2,17 the MtuSSB tetramer is be 2 15 cal/mol per A 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 (A ˚) b (A ˚) c (A ˚) Data resolution (A Unique reflections Completeness (%) (final shell) Rmerge (final shell) Multiplicity (final shell) Rmeasureb (final shell) Phasing powerc Refinement R-factor (Rfree) ˚) Resolution range (A RMS deviation from ideality ˚) Bonds (A 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 A 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† ˚ 2. The buried surface area with a probe radius of 1.4 A 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 A 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 A

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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Structure of M. tuberculosis SSB

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Edited by R. Huber (Received 29 January 2003; received in revised form 23 May 2003; accepted 3 June 2003)