Journal of Molecular Biology
Plasmodium falciparum SSB Tetramer Wraps Single-Stranded DNA with Similar Topology but Opposite Polarity to E. coli SSB
Graphical Abstract
Highlights
► Crystal Structure and DNA binding Properties of Plasmodium falciparum SSB. ► DNA wraps around Pf-SSB with opposite polarity to Ec-SSB. ► Pf-SSB has unique DNA binding properties.
Introduction
Plasmodium falciparum is a eukaryotic parasite and the causative agent for over 250 million cases of malaria that result in 5 million deaths annually.1 It contains a unique non-photosynthetic plastid-like organelle called the apicoplast, which is involved in a variety of biosynthetic pathways of the parasite. A single apicoplast is present in each cell and functions in isoprenoid, fatty acid and heme synthesis/metabolism and is critical to parasite survival and pathogenesis, making it a logical target for antimalarial drugs. The ∼ 35-kb apicoplast genome contains 68 open reading frames that encode a variety of ribosomal proteins, tRNAs, RNA polymerase, chaperones and other proteins of unknown function.2 However, proteins involved in DNA metabolism are encoded by the nuclear DNA and targeted for transport to the apicoplast by an apicoplast localization signal (ALS), which is cleaved upon delivery to the apicoplast.3 The single-stranded DNA binding (SSB) protein from P. falciparum [P. falciparum SSB (Pf-SSB)] is encoded in the nucleus and transported to the apicoplast where it likely functions in the replication and maintenance of the apicoplast genome.4
SSB proteins are present in nearly all organisms and bind to single-stranded DNA (ssDNA) intermediates produced transiently during replication, repair and recombination. Escherichia coli SSB (Ec-SSB) is a well-characterized prototype of bacterial SSB proteins5 and shares high sequence homology with Pf-SSB (39% identity and 66% homology).4 Ec-SSB functions as a homo-tetramer with each subunit consisting of two domains, an N-terminal OB-fold containing the ssDNA binding site and an unstructured C-terminal tail.5, 6, 7 Ec-SSB also interacts with more than a dozen other proteins involved in DNA metabolism.8 These interactions are primarily mediated through a conserved stretch of 8–10 amino acids located at the end of its unstructured C-termini.8
In the case of Ec-SSB, it has been shown that, at moderate to high salt concentrations, an ssDNA ∼ 65 nucleotides long can fully wrap around the tetrameric DNA binding core to form the so-called (SSB)65 binding mode.9, 10, 11 However, due to its four potential DNA binding sites, Ec-SSB can also bind to long ssDNA in a number of different binding modes that differ by the number of subunits (OB-folds) within the tetramer that contact the DNA.10, 12, 13 In the (SSB)65 mode, ssDNA interacts with all four subunits and displays little tendency to form cooperative clusters along ssDNA.14, 15 The low cooperative, fully wrapped (SSB)65 binding mode has been proposed to facilitate RecA-mediated DNA strand exchange during homologous recombination.16, 17, 18 In fact, Ec-SSB, while bound in its (SSB)65 mode, is able to diffuse along ssDNA and transiently melt DNA hairpins, thus facilitating RecA filament formation along ssDNA.18 Here, we present a structural study of the Pf-SSB protein and its complexes with ssDNA including a crystal structure of a Pf-SSB tetramer in complex with ssDNA in a fully wrapped binding mode allowing a detailed comparison of its structure with Ec-SSB.
Section snippets
Pf-SSB forms a stable homo-tetramer in solution
SSB proteins can exist in a variety of oligomeric states including monomers (e.g., T4 phage gp32),19 dimers (e.g., Deinococcus radiodurans SSB),20 trimers (e.g., eukaryotic RPA),21 tetramers (most bacterial SSBs)8 and pentamers (e.g., D. radiodurans DdrB).22 Based on dynamic light scattering and sucrose density gradient analysis, a histidine-tagged version of recombinant Pf-SSB appears to behave as a homo-tetramer in solution.4 Here, we examined the assembly state of an untagged version of Pf
Discussion
We describe a crystal structure of the Pf-SSB tetramer bound to ssDNA at 2.1 Å resolution. All four subunits interact with the ssDNA, and the topology of the DNA path resembles the “seams of a baseball” as observed for Ec-SSB in its fully wrapped (SSB)65 DNA binding mode (Fig. S5).11 Although crystal structures of SSB proteins from multiple organisms have been reported in their apo-form,20, 40, 41, 42, 43, 44, 45 only three SSB–DNA complex structures have been reported.11, 39 Of these, only the
Buffers
Buffer H0.08 is 10 mM Hepes (pH 8.1), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.08 M NaCl and 1 mM TCEP. Buffer H0.2 is 10 mM Hepes (pH 8.1), 0.1 mM Na3EDTA, 200 mM NaCl and 5 mM 2-ME. Lysis buffer is 50 mM Tris–Cl (pH 8.3), 1 mM EDTA, 200 mM NaCl, 10% sucrose and 15 mM spermidine. Buffer Tx is 50 mM Tris–Cl (pH 8.3), 1 mM EDTA and 5% (v/v) glycerol, where “x” denotes the molar concentration of NaCl. Storage buffer is 20 mM Tris–Cl (pH 8.3), 1 mM Na3EDTA, 500 mM NaCl, 5 mM 2-ME and 50%
Acknowledgements
The P. falciparum genomic DNA was a kind gift from Dr. Daniel Goldberg (Washington University School of Medicine). We thank Dr. Alex Kozlov and Dr. Binh Nguyen for significant technical advice and Mr. Thang Ho for synthesis and purification of the oligodeoxynucleotides. This work was supported in part by grants from the National Institutes of Health to T.M.L. (GM30498 and GM45948) and S.K. (GM073837).
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2017, Journal of Molecular BiologyCitation Excerpt :Recently, Kozlov et al. [27] made the surprising finding that the 56 amino acid C-terminal IDL is essential for highly cooperative binding at low salt concentrations. Furthermore, the amino acid composition of the IDL influences cooperative binding as indicated by substitution of the more highly charged IDL from P. falciparum SSB [50,51] for the E. coli IDL [27]. It had been thought that only the (SSB)35 binding mode displayed highly cooperative binding and that the (SSB)65 binding mode showed only limited cooperativity.
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2017, Journal of Biological ChemistryCitation Excerpt :This could be a consequence of the lack of the C-terminal tail in mtSSB. The importance of the C-terminal tail in modulating the binding mode change was reported for PfSSB, which does not show negative cooperativity in dT35 binding mode (23, 24), and the amino acid composition of the C-terminal tail in PfSSB (23) is different from the EcoSSB. Calorimetric measurements of mtSSB binding to dT60 show that the binding enthalpy change (ΔH60obs) is slightly less than the values measured for E. coli SSB binding to dT65 under similar conditions, possibly due to the smaller size of the protein.