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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Reprint requests to: Gabriel Waksman, Institute of Structural Molecular Biology, University College and Birkbeck College, Malet Street, London WC1E 7HX, UK; e-mail: g.waksman{at}mail.cryst.bbk.ac.uk; fax: 44-0207-631-6833.
(RECEIVED January 29, 2004; FINAL REVISION March 31, 2004; ACCEPTED March 31, 2004)
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Keywords: SSB; crystal structure; DNA replication; ssDNA, protein disorder
1 Present addresses: Department of Biochemistry, Physiology and Microbiology, Ghent University, 9000 Ghent, Belgium; 
2 The University of Texas Southwestern Medical Center at Dallas, Dallas TX 75390, USA;
3 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK;
4 Institute of Structural Molecular Biology, University College and Birkbeck College, London WC1E 7HX, UK.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04661904.
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Introduction |
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Recent structural analyses of a chymotryptic fragment of E. coli SSB (SSBc) containing the ssDNA-binding domain of the protein (residues 1–135), which still forms a tetramer, have provided a detailed view of the structural determinants underlying its interactions with ssDNA (Ragunathan et al. 1997, 2000; Yang et al. 1997). These studies revealed the details of the topology for the wrapping of ssDNA around the tetrameric SSBc scaffold, and the fact that consecutive SSBc tetramers participate in crystal packing interactions via their respective L45 loops (loops between the 
4 and 5 strands in the SSBc OB fold), resulting in linear SSBc polymers. Taken together, these observations suggest that the interactions mediated by the L45 loops may play a role in facilitating the unlimited positive cooperativity observed for the (SSB)35 binding mode.
In addition to the interactions of SSB with ssDNA, a second essential aspect of its function is its interaction with proteins involved in DNA metabolism. So far, a number of proteins have been demonstrated to interact with SSB: exonuclease I (Genschel et al. 2000), RecO (Umezu and Kolodner 1994; Kantake et al. 2002), uracil DNA glycosylase (Handa et al. 2001), and the 
subunit of DNA polymerase III (pol III) holoenzyme (Glover and McHenry 1998; Kelman et al. 1998; Witte et al. 2003). An important consensus that has emerged from these studies is that SSB uses its C-terminal domain to interact with these protein partners. More detailed studies of the binding of SSB to the subunit of the pol III holoenzyme have shown that the cornerstone of this interaction lies within the last 26 amino acid residues from the C terminus of SSB (Kelman et al. 1998; Witte et al. 2003).
Although the structure and interactions of the DNA-binding domain of SSB have been described in detail (Ragunathan et al. 1997, 2000; Yang et al. 1997), the structure of the C-terminal domain (residues 113–177) has remained elusive. Here, we attempt to shed light onto the structural features of full-length SSB and how these may relate to its function.
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).
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; Table 1). The core DNA-binding domain of SSB could be readily modeled in the experimental electron density maps up to residue 112 in each molecule, and it is almost identical to the structure of SSBc with and without ssDNA (Ragunathan et al. 1997, 2000). On the other hand, the electron density corresponding to the C-terminal domain was undecipherable, suggesting high mobility in this part of the structure. In contrast to previous studies of SSBc in complex with dC(pC)34 (Ragunathan et al. 2000), the electron density for ssDNA was generally poor and discontinuous, and only well defined where the DNA bases are involved in stacking interactions with tryptophan residues of SSB (Ragunathan et al. 2000). Convincing modeling of the ssDNA was therefore not possible. Although the effective resolution of our analysis is a limiting 3.3 Å, the sharp contrast in electron density quality between the disordered C-terminal domain and the well-ordered core domain strongly suggests that our interpretation is qualitatively correct. Furthermore, the low-resolution diffraction of SSB-dC(pC)34 crystals is consistent with the observed molecular disorder and with the consensus that macromolecules with high degrees of flexibility and/or lack of regular structure either fail to crystallize or yield weakly diffracting crystals. Iterative cycles of fourfold noncrystallographic symmetry averaging and solvent flipping improved the electron density for the DNA-binding core, but not the missing C-terminal regions, suggesting that the C-terminal domains adopt several distinct conformations and/or that they are largely unstructured. The absence of specific interactions of the C-terminal regions with the core of the protein, and the presence of pronounced bulk solvent pockets next to the DNA-binding domains suggest that the disordered/unstructured C-terminal regions may emanate laterally away from the core of the structure (Fig. 2B). At the same time we observe that adjacent SSB tetramers within the crystal interact via their L45 loops to form a "filament" of protein molecules (Fig. 2C). Such interactions have previously been observed in two different crystal forms of SSBc (Ragunathan et al. 1997, 2000). The fact that we observe this same interaction in a third distinct crystal lattice strongly suggests that this may be a general property of SSB and that it may form the structural basis for cooperative binding of SSB in its (SSB)35 binding mode.
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The low sequence complexity of the C-terminal domain of SSB (heavily populated by proline, glycine, and glutamine residues) had earlier hinted for the possibility of high disorder and lack of structure (Sancar et al. 1981). Indeed, predictions of disorder carried out by using disEMBL (http://dis.embl.de) Linding et al. 2003) identified the entire C-terminal domain as potentially unstructured/disordered. Furthermore, alignment of SSB sequences from diverse sources revealed the presence of a conserved acidic tail at the end of the C-terminal domains (Kelman et al. 1998). Interestingly, this region forms a C-terminal 
-helix in the crystal structure of the phage T4 gene 32 protein and was found to lie on the surface of the molecule, suggesting that interactions with partner proteins take place in close proximity to the core of the DNA-binding domain (Shamoo et al. 1995). The absence of such a structural feature in E. coli SSB suggests that interactions of SSB with proteins of the replication machinery, such as the subunit of Pol III holoenzyme, may take place away from the DNA-binding core of the protein. The C terminus of SSB contains the negatively charged consensus sequence DDDIPF, which may act as the tether-point onto complementary positively charged surfaces on the subunit. Indeed, the recently published structure of the –
heterodimeric complex of E. coli DNA Polymerase III has revealed a conserved, positively charged patch defined by residues 124–135 on the surface of the subunit at the distal end of the – complex (Gulbis et al. 2004). It was therefore proposed that this may be the docking site for the C terminus of SSB (Gulbis et al. 2004).
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Materials and methods |
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-mercapto-ethanol at 20°C and then diluted 100-fold in 100 mM Tris HCl (pH 8.5), 1 mM EDTA, and 1 mM -mercapto-ethanol to adjust the NaCl concentration to 5 mM. The complex was then concentrated to ~10 mg/mL. Crystals were grown in a hanging drop against a reservoir containing a solution of 100 mM Hepes (pH 7.5), 20% (v/v) PEG200, and 20% (w/v) PEG4000. Sizable crystals (0.3 x 0.3 x 0.3 mm) of SSB–dC(pC)34 appeared reproducibly within 2 to 3 months. Crystals of SSB–dC(pC)34 belong to space group P3112 with a = b = 60.9 Å, c = 348.9 Å, = = 90°, 
= 120° and a tetramer in the asymmetric unit.
Characterization of SSB–dC(pC)34 crystals
SSB–dC(pC)34 crystals were tested for both the presence of DNA and the integrity of the protein. To assess the integrity of the protein, crystals were first washed by using the same procedure as above and then dissolved in an SDS-PAGE loading buffer, and the protein was visualized on a Coomassie blue–stained 12% SDS-PAGE gel (Fig. 1A
). To test for the presence of DNA, the crystals of SSB–dC(pC)34 were first repeatedly washed in a stabilizing solution containing 100 mM Hepes (pH 7.5), 20% (v/v) PEG200, and 20% (w/v) PEG4000 and then dissolved in a standard Trisborate-EDTA (TBE) buffer containing 6 M urea and 10% glycerol. The sample was then loaded onto a 19% polyacrylamide slab gel. After electrophoresis for 3 h at 5 to 10 W (constant power), the gel was stained by using a solution of 0.01% (w/v) stains-all (Aldrich) and 40% (v/v) formamide (Fig. 1B).
Structure determination
Complete data sets to 3.3 Å resolution were collected at three wavelengths (SeMet-1 through -3; Table 1) at beam line 19ID of the Structural Biology Center, Advanced Photon Source, and were processed with DENZO and SCALEPACK (Otwinowski and Minor 1997). Automated approaches in obtaining phasing information by MAD or single-wavelength anomalous diffraction (SAD) using CNS (Brünger et al. 1998) and SOLVE (Terwilliger and Beredzen 1999) failed due to the fact that the selenium substructure could not be determined. The following strategy was then successfully used to obtain de novo phasing information.
A molecular replacement solution for a tetramer in the asymmetric unit was obtained by using the structure of the chymotryptic fragment of SSB (PDB code 1EYG
[PDB]
; Raghunathan et al. 2000). This was then combined with data collected at the absorption peak of selenium (SeMet-3; Table 1), to produce an anomalous difference Patterson map that revealed 12 selenium sites in the core domain. The additional selenium site expected at the end of the C-terminal domain could not be located in any of the four SSBfl molecules indicative of local and/or domain disorder. Introduction of the derived selenium substructure into MAD phasing protocols in CNS and SOLVE using data from all three wavelengths failed to produce an interpretable electron density. On the other hand, SAD phasing in CNS with either data collected at the selenium absorption peak (SeMet-3; Table 1) or the selenium absorption edge (SeMet-2; Table 1) followed by electron density modification by solvent flipping yielded a partially interpretable electron density map. Surprisingly, a greatly improved electron density map could be obtained by MAD phasing in CNS using both SeMet-2 and SeMet-3, followed by solvent flipping (Fig. 2A). The core domain of SSB could be readily modeled in the experimental electron density maps up to residue 112 in each molecule, whereas the electron density of the anticipated C-terminal domain was undecipherable suggesting extreme disorder. The electron density for ssDNA was also poor, and only well defined where bases are involved in stacking interactions with tryptophan residues of SSB. Iterative cycles of fourfold noncrystallographic symmetry averaging and solvent flipping improved the electron density for the DNA-binding core but not the missing C-terminal regions, suggesting that the C-terminal domains adopt distinct conformations. Modeling of the SSB beyond residue 112 and of ssDNA was therefore not possible. Initially, model building was carried out by docking the DNA-binding domain of SSB (Raghunathan et al. 2000) into experimental electron density maps by using the program O (Jones et al. 1991). Crystallographic refinement was carried out by using rigid-body refinement and conjugate gradient minimization in CNS, followed by grouped-temperature factor refinement. Heavy NCS-restraints were initially applied evenly to all four protomers but were subsequently optimized for loop regions by using the behavior of the Rfree value as a guide.
Crystallographic coordinates and structure factors have been deposited with the Protein Data Bank (http://www.rcsb.org) with accession code 1SRU and will be available upon the publication date of this article.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Bujalowski, W. and Lohman, T.M. 1986. Escherichia coli single-stranded DNA-binding protein forms multiple, distinct complexes with single-stranded DNA. Biochemistry 25: 7799–7802.[CrossRef][Medline]
Chase, J.W. and Williams, K.R. 1986. Single-stranded DNA binding proteins required for DNA replication. Annu. Rev. Biochem. 55: 103–136.[CrossRef][Medline]
Chrysogelos, S. and Griffith, J. 1982. Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proc. Natl. Acad. Sci. 79: 5803–5807.
Genschel, J., Curth, U., and Urbanke, C. 2000. Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. Biol. Chem. 381: 183–192.[CrossRef][Medline]
Glover, B.P. and McHenry, C.S. 1998. The subunits of DNA polymerase III holoenzyme bind to single-stranded DNA-binding protein (SSB) and facilitate replication of an SSB-coated template. J. Biol. Chem. 273: 23476–23484.
Griffith, J.D., Harris, L.D., and Register, J. 1984. Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments. Cold Spring Harb. Symp. Quant. Biol. 49: 553–559.[Medline]
Gulbis, J.M., Kazmirski, S.L., Finkelstein, J., Kelman, Z., O’Donnell, M., and Kuriyan, J. 2004. Crystal structure of the: sub-assembly of the Escherichia coli DNA polymerase clamp-loader complex. Eur. J. Biochem. 271: 439–449.[Medline]
Handa, P., Acharya, N., and Varshney, U. 2001. Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Myobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases. J. Biol. Chem. 276: 16992–16997.
Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47: 110–119.
Kantake, N., Madiraju, M.V.V.M., Sugiyama, T., and Kowalczykowski, S.C. 2002. Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination. Proc. Natl. Acad. Sci. 99: 15327–15332.
Kelman, Z., Yuzhakov, A., Andjelkovic, J., and O’Donnell, M. 1998. Devoted to the lagging strand—the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly. EMBO J. 17: 2436–2449.[CrossRef][Medline]
Linding, R., Jensen, L.J., Diella, F., Bork, P., Gibson, T.J., and Russell, R.B. 2003. Protein disorder prediction: Implications for structural proteomics. Structure 11: 1453–1459.[Medline]
Lohman, T.M. and Ferrari, M.E. 1994. Escherichia coli single stranded DNA-binding protein: Multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem. 63: 527–570.[Medline]
Lohman, T.M. and Overman, L.B. 1985. Two binding modes in Escherichia coli single strand binding protein–single stranded DNA complexes: Modulation by NaCl concentration. J. Biol. Chem. 260: 3594–3603.
Lohman, T.M., Green, J.M., and Beyer, R.S. 1986a. Large-scale production and rapid purification of the Escherichia coli gene product: Expression of the ssb gene under 
PL control. Biochemistry 25: 21–25.[CrossRef][Medline]
Lohman, T.M., Overman, L.B., and Datta, S. 1986b. Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein. J. Mol. Biol. 187: 603–615.[CrossRef][Medline]
Meyer, R.R. and Laine, P.S. 1990. The single-stranded DNA-binding protein of Escherichia coli. Microbiol. Rev. 54: 342–380.
Ollis, D., Brick, P., Abdel-Meguid, S.S., Murthy, K., Chase, J.W., and Steitz, T.A. 1983. Crystals of Escherichia coli single-strand DNA-binding protein show that the tetramer has D2 symmetry. J. Mol. Biol. 170: 797–800.[Medline]
Otwinowski, Z. and Minor, W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307–326.
Raghunathan, S., Ricard, C.S., Lohman, T.M., and Waksman, G. 1997. Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9 Å resolution. Proc. Natl. Acad. Sci. 94: 6652–6657.
Raghunathan, S., Kozlov, A.G., Lohman, T.M., and Waksman, G. 2000. Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nature Struct. Biol. 7: 648–652.[CrossRef][Medline]
Sancar, A., Williams, K.R., Chase, J.W., and Rupp, W.D. 1981. Sequences of the ssb gene and protein. Proc. Natl. Acad. Sci. 78: 4274–4278.
Shamoo, Y., Friedman, A., Parsons, M.R., Koningsberg, W.H., and Steitz, T.A. 1995. Crystal structure of a replication fork single-stranded DNA binding protein (T4 gp32) complexed to DNA. Nature 376: 362–366.[CrossRef][Medline]
Terwilliger, T.C. and Berendzen, J. 1999. Automated MAD and MIR structure solution. Acta Crystallogr. D 55: 849–861.[CrossRef][Medline]
Tompa, P. 2002. Instrinsically unstructured proteins. Trends Biochem. Sci. 27: 527–533.[CrossRef][Medline]
Umezu, K. and Kolodner, R.D. 1994. Protein interaction in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single stranded DNA-binding protein. J. Biol. Chem. 269: 30005–30013.
Witte, G., Urbanke, C., and Curth, U. 2003. DNA polymerase III subunit ties single-stranded DNA binding protein to the bacterial replication machinery. Nucleic Acids Res. 31: 4434–4440.
Wong, I., Chao, K.L., Bujalowski, W., and Lohman, T.M. 1992. DNA-induced dimerization of the Escherichia coli rep helicase: Allosteric effects of single-stranded and duplex DNA. J. Biol. Chem. 267: 7596–7610.
Wright, P.E. and Dyson, H.J. 1999. Intrinsically unstructured proteins: Reassessing the protein structure-function paradigm. J. Mol. Biol. 293: 321–331.[CrossRef][Medline]
Yang, W., Hendrickson, W.A., Kalman, E.T., and Crouch, R.J. 1990. Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli. J. Biol. Chem. 265: 13533–13559.
Yang, C., Curth, U., Urbanke, C., and Kang, C. 1997. Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 Å resolution. Nat. Struct. Biol. 4: 153–157.[CrossRef][Medline]
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. The final refined model is shown as a reference in stick representation color-coded in yellow for carbon, blue for nitrogen, and red for oxygen. (B) A view of the crystal packing of the SSB tetramer. The protomer is drawn in cyan and is surrounded by symmetry related molecules. The white asterisks indicate the points (Leu 112) in each of the four subunits in the SSB tetramer at which interpretable experimental electron density stops. The horizontal dyad axis is indicated as a white dotted line as a reference, and the cavities in the packing accommodating the disordered C-terminal regions are shown in red circles. (C) Conservation of the L45-mediated interactions of successive SSB molecules to produce a string polymer of SSB molecules. This view is obtained by rotating the one in B by 90 degrees counterclockwise around the vertical axis. The L45 mediated interactions are encircled in dotted circles.