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Structural insight into the length-dependent binding of ssDNA by SP_0782 from Streptococcus pneumoniae, reveals a divergence in the DNA-binding interface of PC4-like proteins

Abstract SP_0782 from Streptococcus pneumoniae is a dimeric protein that potentially binds with single-stranded DNA (ssDNA) in a manner similar to human PC4, the prototype of PC4-like proteins, which plays roles in transcription and maintenance of genome stability. In a previous NMR study, SP_0782 exhibited an ssDNA-binding property different from YdbC, a prokaryotic PC4-like protein from Lactococcus lactis, but the underlying mechanism remains unclear. Here, we show that although SP_0782 adopts an overall fold similar to those of PC4 and YdbC, the ssDNA length occupied by SP_0782 is shorter than those occupied by PC4 and YdbC. SP_0782 exhibits varied binding patterns for different lengths of ssDNA, and tends to form large complexes with ssDNA in a potential high-density binding manner. The structures of SP_0782 complexed with different ssDNAs reveal that the varied binding patterns are associated with distinct capture of nucleotides in two major DNA-binding regions of SP_0782. Moreover, a comparison of known structures of PC4-like proteins complexed with ssDNA reveals a divergence in the binding interface between prokaryotic and eukaryotic PC4-like proteins. This study provides insights into the ssDNA-binding mechanism of PC4-like proteins, and benefits further study regarding the biological function of SP_0782, probably in DNA protection and natural transformation.


INTRODUCTION
In order to maintain the integrity of genetic information, cells employ many types of single-stranded DNA binding proteins (SSBs) to protect single-stranded DNA (ssDNA) from damage or to recruit repair proteins to the DNA damage site (1)(2)(3). The various ssDNAbinding domains engaged by different SSBs can be distinguished according to their structures. The well-studied ssDNA-binding domains are structurally characterized as oligosaccharide/oligonucleotide-binding (OB) folds (4), K homology (KH) domains (5), RNA recognition motifs (RRMs) (6) and whirly domains (7). OB folds usually consist of a five-stranded ␤-sheet and one ␣-helix (4), and assemble into multimers, such as the well-known homotetramer formed by the SSB from Escherichia coli (EcSSB) (8). There are also OB fold-derived structures that form homodimers to bind with ssDNA, such as those from the positive cofactor 4 (PC4)/Sub1-like proteins (9). PC4/Sub1 was initially identified as a transcriptional coactivator, and later on its roles in chromatin condensation, maintenance of genome stability and DNA repair were implicated (reviewed in (3)). The C-terminal domain (CTD) of human PC4 is an ssDNA-binding domain that can dimerize to bind with ssDNA or melted double-stranded DNA (dsDNA) in a sequence-independent manner (10,11). In a PC4-CTD dimer, each subunit consists of one ␣-helix and a four-stranded ␤-sheet that forms a concave for grasping five nucleotides in one strand of DNA. Two subunits cooperate in binding with each of two opposing DNA strands through hydrogen bonds, hydrophobic interaction and electric charges (9). The ssDNA bound by PC4 should be long enough to bend into two antiparallel strands, as PC4 shows low binding affinity to ssDNA shorter than 10 nt (10). On the other hand, for ssDNA with a length of 28 nt, two dimers of PC4 will bind with it (12,13). PC4 is evolutionarily conserved and present in other eukaryotes, including yeast and Magnaporthe oryzae, as well as in prokaryotes and Bacteriophage T5 (14)(15)(16)(17). Its homologs MoSub1 in M. oryzae, YdbC in Lactococcus lactis, SSB in Bacteriophage T5, and BPSL1147 in Burkholderia pseudomallei, all exhibit a quaternary structure of the homodimer (15)(16)(17)(18). The binding property of YdbC with ssDNA dT19G1 is similar to that of human PC4-CTD, and it can bind with ssDNA of 20 nt in a two-dimer-bound form, although the affinity of the second dimer is much weaker (16). Homolog searching with YdbC identified hundreds of prokaryotic PC4-like proteins with unknown structure and function, which may have distinct ssDNA-binding properties. In fact, EF 3132 from Enterococcus faecalis shows dynamics of conformational exchange between DNA-free and DNA-bound states significantly different from that of YdbC (16). Thus, the structure and function of prokaryotic PC4-like proteins is a topic that demands further broad and deep exploration.
Streptococcus pneumoniae is a Gram-positive and significant human pathogenic bacterium. There are two homologs of EcSSB assembling into homotetramers but showing different ssDNA-binding properties in S. pneumoniae, designated as SpSsbA and SpSsbB (19). However, the potential homolog of PC4 and YdbC was not well characterized. We previously reported an NMR study on SP 0782 from S. pneumoniae, which is 56.2% identical to YdbC in sequence (20). SP 0782 exists as a homodimer in solution, similar to PC4-CTD and YdbC. However, it exhibited a different binding property toward the ssDNA dT19G1 in NMR titration experiments compared to YdbC. To better understand the underlying mechanism for the different binding property, in this study, we investigated the ssDNA binding of SP 0782 in detail, and determined the structures of apo SP 0782 and SP 0782:ssDNA complexes, which reveal a distinctive ssDNA-binding mechanism of SP 0782 from those of PC4 and YdbC, and suggest a variation of PC4-like proteins in the ssDNA-binding interface. Moreover, the DNA-binding patterns of SP 0782 for different lengths of ssDNA were revealed, enlightening the mechanism by which SP 0782 assembles at the exposed singlestrand regions of genomic DNA that are variable in length.

NMR structure calculation and NMR titration
All NMR data for structure calculation were collected at 293 K using 600 MHz Varian Inova and 850 MHz Bruker Avance NMR spectrometers equipped with 5-mm cryogenic probes, processed using NMRPipe and analyzed with Sparky program. Backbone and side chain resonance assignments were described previously (20) and deposited in the BioMagResBank (BMRB) with an accession number 17175. NOE-based inter-proton distance restraints were determined automatically using CYANA 2.1. Input for CYANA consisted of chemical shift assignments, NOESY peak lists from three NOESY spectra with peak intensities, the restraints for backbone phi () and psi () torsion angles derived from chemical shifts of backbone atoms using the TALOS+ software program (21). Manual and iterative refinements of NOESY peak picking lists were guided using NMR RPF quality to assess 'goodness of fit' between calculated structures and NOESY peak lists (22). The 20 lowest energy structures calculated by CYANA 2.1 were further refined using restrained molecular dynamics in explicit water CNS 1.2 (23) and the PARAM19 force field, using the final NOE-derived distance constraints and TALOS-derived dihedral angle restraints. The final NMR ensemble of 20 structures was deposited in the Protein Data Bank (PDB, ID: 2L3A). Structural statistics and global structure quality factors were computed using PSVS version 1.5 (24) (Supplementary Table S1). NMR titrations were performed on a 600 MHz Bruker spectrometer at 293 K. The samples containing 0.4 mM SP 0782 and different concentrations of dT6, dT12 or dT19G1 were pre-mixed and allowed to equilibrate for 1 h, and subsequently used for 1 H-15 N HSQC spectrum collection. Chemical shift perturbations (CSPs) were calculated using the equation proposed in a previous study (25).

Isothermal titration calorimetry (ITC)
ITC was performed on a VP-ITC instrument (GE Healthcare) at 293 K. All samples were prepared in the same buffer as that used in the NMR experiments to avoid any heat change resulted from mixing buffers. Solutions containing 100 M of dT6, dT12 or dT19G1 were titrated into the reservoir containing 10 M of SP 0782, respectively. Each titration experiment consisted of a preliminary injection of 2 l followed by 27 injections of 10 l, with a duration of 20 s and a delay of 180 s between each injection. Thermogram analysis was performed using MicroCal Origin 7.0 (Microcal Software Inc.) with a one-site binding model. The first data point was not included when the titration curves were fitted. Dilution heat was subtracted with a ctrl titration through injecting the DNA solution into buffer solution alone for each experiment. The ITC experiments were repeated in triplicate.

Electrophoretic mobility shift assay (EMSA)
EMSA experiments between SP 0782 and ssDNA probes with biotin label at 5 -end were carried out using a Chemiluminescent EMSA Kit (Beyotime Biotech Co.). The samples containing 0.1 pmol ssDNA probes (final concentration of 0.01 M in a 10 l binding mixture) and different contents of SP 0782 were premixed and incubated for 25 min at room temperature, and subsequently loaded into a 10% native PAGE gel in 0.5× TBE buffer. After separation by electrophoresis, the free probe and the complex of probe and protein were transferred onto a nylon membrane and subjected to UV crosslinking. Subsequently detection of chemiluminiscence was conducted following the protocol of the Chemiluminescent EMSA Kit, and using a Chemi-Doc MP instrument (Bio-rad).

Protein-DNA complex crystallization and structure determination
Octahedron-shaped crystals of four SP 0782:ssDNA complexes were obtained by sitting drop vapor diffusion. Briefly, lyophilized SP 0782 was dissolved in a crystallization buffer (5 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM DTT and 0.02% (w/v) NaN 3 ) to reach a final protein concentration of 8 mg/ml with ssDNA (dTCTTCC, dT6 or dT12) supplied at a 2:1 molar ratio to protein, and incubated for about 1 h on ice before crystallization. 0.4 l of each protein-DNA mixture was mixed with an equal volume of a precipitant solution. Crystals appeared after 1 week in precipitant solutions of 0.2 M potassium nitrate and 20% (w/v) polyethylene glycol (PEG) 3350 for SP 0782:dTCTTCC, 0.2 M sodium nitrate, pH 6.8 and 20% (w/v) PEG3350 for SP 0782:dT6, and 0.07 M citric acid, 0.03 M Bis-Tris propane, pH 3.4, and 16% (w/v) PEG3350 for SP 0782:dT6 and SP 0782:dT12. Crystals were transferred to cryo-solutions (precipitant solutions supplemented with 20% (v/v) glycerol) through incremental buffer exchange prior to flash cooling in liquid nitrogen.
Single crystal X-ray diffraction data were collected at 100 K on Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 for SP 0782:dTCTTCC, and BL19U1 for SP 0782:dT6 and SP 0782:dT12. At least 180 • of data were typically collected in 0.2-0.5 • oscillation steps. The data were indexed, merged and scaled with HKL2000 (26). The initial structure solution was obtained using the molecular replacement program PHASER (27) with an apo SP 0782 structure (PDB ID: 3OBH) as the search model. Further model building and structure refinement were done using Coot and Phenix (28,29), respectively. 5000 K composite simulated-annealing (SA) omit 2F o -F c electron density maps were generated using the Cartesian-Anneal method (30) by Phenix. All structures were superposed using the maximum likelihood based superpositioning program THESEUS (31) unless otherwise indicated. Data collection and refinement statistics are summarized in (Supplementary Table S2) and the atomic coordinates and structure factors have been deposited in the PDB (PDB ID: 5ZKL (SP0782:dT12), 5ZKM (SP0782:dTCTTCC), 6JIQ (SP 0782:dT6, form 1) and 6JIP (SP 0782:dT6, form 2)).

Solution structure of apo SP 0782
Our previous work revealed that apo SP 0782 exists predominantly as a homodimer in solution under the used conditions, suitable for 3D structure determination using NMR spectroscopy (20). We report here the solution NMR structure of apo SP 0782. Each subunit of dimeric SP 0782 contains a curved anti-parallel ␤-sheet composed of four ␤-strands in the N-terminus and one 12-residue ␣-helix in the C-terminus ( Figure 1A and Supplementary Figure  S1A). All secondary structural elements are well-defined as follows: ␤1 (F14-N26), ␤2 (W30-F39), ␤3 (A44-W50), ␤4 (I62-L64), and ␣1 (N66-K77). The two domain-swapped helices arrange with an angle of ∼60 • to each other, and pack mainly against the ␤-sheet of their respective dimeric partner. To date, the crystal structure of SP 0782 (PDB ID: 3OBH) was also solved by scientists from the Northeast Structural Genomics (NESG) consortium ( Figure 1B) (32). By comparison, the two structures share highly similar overall folds, except that ␤4 (M58-L64) in the crystal structure is a little longer than its counterpart in the solution NMR structure, which can be reasonably explained by insufficient observed NOE peaks in this region due to relatively flexible conformational dynamics in solution ( Figure 1C).
Sequence alignment shows that SP 0782 shares high similarity to YdbC and EF 3132, two prokaryotic homologs with determined structures, with a sequence identity of 56.2% and 53.4%, respectively (Supplementary Figure S1B). As expected, three proteins exhibit high structural similarity (Supplementary Figure S1C). ConSurf (33) analysis shows that highly conserved residues are mostly located Nucleic Acids Research, 2020, Vol. 48, No. 1 435 on the antiparallel ␤-sheet and the loops connecting the ␤strands, except for E68 in the ␣-helix ( Supplementary Figure S2A). Electrostatic surface potential analysis (34) shows that the ␤-sheet surface consists of many positively charged amino acids, whereas the backside is made up of negatively charged amino acids (Supplementary Figure S2B). Seven positively charged residues located at the ␤-sheet, including K28, K32, R36, K45, R49, K57 and K60, extend their side chains to participate in forming one face of the SP 0782 dimer with extensively positive charge, similar to those of PC4-CTD and YdbC. In brief, SP 0782 exhibits a PC4-like fold potential for ssDNA binding.

The varied binding patterns of SP 0782 for different lengths of ssDNA
The binding of both YdbC and PC4-CTD with the ss-DNA dT19G1 has been well studied (9,16), while the binding of SP 0782 with dT19G1 has previously been preliminarily investigated through NMR titration, which showed that most peaks of SP 0782 disappeared during dT19G1 titration (Supplementary Figure S3) (20). Further NMR experiments at different temperatures revealed that increasing temperature could significantly enhance the peak intensities of SP 0782 titrated with dT19G1 at protein (monomer):DNA ratios of 2:1.2 and 2:0.2, but the observed peaks at the protein:DNA ratio of 2:0.2 were much less than those at the protein:DNA ratio of 2:1.2 (Supplementary Figure S4). As increasing temperature causes more rapid tumbling and may turn the conformational exchange at intermediate rate on the NMR timescale into fast rate (35,36), the peak disappearance of SP 0782 at the protein:DNA ratio of 2:1.2 should mainly result from a conformational exchange between apo and dT19G1-bound states at intermediate rate. On the other hand, at the protein:DNA ratio of 2:0.2, other factors, such as the formation of a high molecular weight (MW) complex, may also contribute to the peak disappearance. In either case, it prevented further NMR study regarding dT19G1 binding. Thus, ssDNA samples other than dT19G1 were employed in NMR titration experiments, wherein dT6 and dT12 that are shorter than dT19G1 did not cause obvious peak disappearance but significant chemical shift perturbations (CSPs) of SP 0782 (Supplementary Figure S3). The binding of SP 0782 was basically saturated at protein(monomer):DNA ratios of 2:2 for dT6 and 2:1 for dT12, respectively. These results confirmed the ssDNA-binding function of SP 0782, and implied varied binding properties of SP 0782 when binding to different lengths of ssDNA.
Since SP 0782 showed different binding properties for dT6, dT12 and dT19G1 in NMR titrations, EMSA experiments were further employed for comparing the binding of SP 0782 with the three ssDNAs. The results showed significantly different binding patterns of SP 0782 for the three ssDNAs ( Figure 2). Two kinds of SP 0782:dT12 complexes with different MWs were detected, with the small one formed at low protein:DNA concentration ratios and the large one formed at high protein:DNA ratios. SP 0782 could bind to dT6, forming a complex with a MW similar to that of the small SP 0782:dT12 complex, while the SP 0782:dT6 complex with a MW similar to that of the large SP 0782:dT12 complex was not steadily detected. In contrast, SP 0782 bound to dT19G1, forming a series of complexes with MWs around that of the large SP 0782:dT12 complex, while no SP 0782:dT19G1 complex with a MW similar to that of the small SP 0782:dT12 complex was detected. Moreover, as SP 0782 concentration increased, the MW of the SP 0782:dT19G1 complex increased stepwise, suggesting the formation of various complexes containing different numbers of SP 0782 dimers ( Figure 2). Additionally, SP 0782 showed obviously different affinities for dT6 and dT12, as revealed by the concentrations of SP 0782 when the SP 0782:ssDNA complexes were initially detected ( Figure 2).
In order to further determine the affinities of SP 0782 for dT6, dT12 and dT19G1 and the stoichiometries in the complexes of SP 0782 with the three ssDNAs, ITC experiments were carried out, and the results are shown in Figure 3 and Table 1. The dissociation constants (K D ) of SP 0782 for dT6, dT12 and dT19G1 are 2.68 × 10 −5 , 2.06 × 10 −8 and 1.69 × 10 −7 M, respectively. The binding affinity for dT6 is much weaker than that for dT12, while the affinity for dT19G1 is also 8.2-fold weaker than that for dT12. The binding stoichiometries of one subunit of the SP 0782 dimer for dT6 and dT12 are 0.81 and 0.49, suggesting that one SP 0782 dimer binds to two dT6 molecules but only binds to one dT12 molecule, consistent with the NMR titration data. Considering the largely higher affinity of SP 0782 for dT12 than dT6, this implies that two subunits in the SP 0782 dimer cooperate to achieve high-affinity binding of ssDNA, similar to PC4-CTD. The stoichiometry of one subunit of the SP 0782 dimer for dT19G1 is close to 3:1 (0.33), suggesting that two dimers bind with one molecule of dT19G1, but among the four subunits of the two dimers, one subunit may not bind with DNA. This may be the reason for the lower affinity of SP 0782 for dT19G1 than dT12. A multi-angle static light scattering assay coupled with size exclusion chromatography (SEC-MALS) showed that the SP 0782:dT19G1 complex containing two SP 0782 dimers and one dT19G1 could steadily exist at the protein:DNA ratios of 2:0.2 and 2:0.1, under conditions similar to those for NMR titrations (Supplementary Figure S5). Thus, based on the EMSA, ITC and SEC-MALS data, one dT19G1 can be bound by two or more SP 0782 dimers at a high protein:DNA ratio, which may lead to a high-density binding. Accordingly, the small SP 0782:dT12 and SP 0782:dT6 complexes detected in EMSA experiments should contain one SP 0782 dimer, while the large SP 0782:dT12 complexes may contain two SP 0782 dimers. To sum up, one SP 0782 dimer could sufficiently bind to ssDNA with a length of ∼12 nt in a high-affinity manner, while at a high protein:DNA ratio, more than one SP 0782 dimer could bind to dT12 and dT19G1, possibly in a high-density manner but with relatively lower affinity.

The ssDNA-binding interface of SP 0782 derived from NMR titrations
The NMR titration data were further analyzed to characterize the interface of SP 0782 for binding with ssDNA, which could be in general derived from changes in peak intensity and position. Figure 4A shows an overlay of 1 H-   Table 1. The ITC profiles shown in Figure 3 were fitted with a one-site binding model and independent sets of binding sites. All parameters were allowed to float during the fitting routines. Values for G and S were calculated using the standard formalisms containing the maximum errors as carried through the equations. 15 N HSQC spectra of SP 0782 titrated with dT12 at a series of monomeric protein:ssDNA ratios. CSPs are clearly observed, and in terms of conformational exchange dynamics on the NMR timescale, three distinct kinds of CSPs were found, indicating residues undergoing fast exchange such as G41, slow exchange such as S65, and intermediate exchange such as T63 ( Figure 4A). The CSP of each cross peak was further analysed, as illustrated in Figure 4B. The residues with CSPs greater than mean + 1, plus another six residues (R49, A50, M58, G59, K60 and T63, labeled with asterisks) showing disappeared signals, were selected to define the ss-DNA binding interface. When mapped onto the solution structure of apo SP 0782, the dT12 binding interface was found to be mainly located on the ␤-sheet ( Figure 4C). In addition, eight residues (T13, K32, V37, E39, N40, I48, I62 and L64) involved in dT12 binding undergo slow conformational exchange, consistent with the nano-molar affinity for dT12 determined in the ITC experiment.
In the case of dT6 titration, most residues with CSPs undergo fast conformational exchange upon dT6 binding (Supplementary Figure S6), consistent with the micromolar affinity of SP 0782 for dT6. The interface of SP 0782 All residues with CSPs greater than 'mean + 1', together with six disappeared residues labelled with asterisks, are further mapped onto the NMR structure of SP 0782 as shown in (C). Two residues undergoing fast exchange are colored in blue, eight residues undergoing slow exchange are colored in red, and six residues undergoing intermediate exchange are colored in green in both (B) and (C).
for dT6 was derived using the NMR titration data following the same process as that for dT12, and is nearly identical to the interface for dT12 (Supplementary Figure S6C). These results indicate that SP 0782 binds with dT6 and dT12 with very different affinities but similar binding interfaces, and support the proposal that the two subunits of the SP 0782 dimer cooperate to bind long ssDNA and achieve highaffinity binding. The binding interface for dT19G1 could not be derived due to the peak disappearance of SP 0782 in NMR titration.

Crystal structures of SP 0782:ssDNA complexes
In order to further dissect the ssDNA-binding mechanism of SP 0782, we determined four crystal structures of SP 0782 in complex with ssDNA ( Figure 5 and Supplementary Figure S7, Table S2). Among these structures, two of them were derived from a SP 0782:dT6 complex with ssDNA resolved to different extent (form 1: two nucleotides; form 2: three nucleotides), and the third one was derived from a SP 0782:dT12 complex. The crystal structure of SP 0782 with dT19G1 that was reported previously (PDB ID: 4G06) (37) was used for comparison with the crystal structures of apo SP 0782, SP 0782:dT6 (both forms), and SP 0782:dT12 ( Figure 5A-E). Compared with apo SP 0782 (PDB ID: 3OBH, chain A), the RMSD values of ssDNA-bound SP 0782 are 0.30, 0.32, 0.38 and 0.94Å for SP 0782:dT6 (form 1), SP 0782:dT6 (form 2), SP 0782:dT12, and SP 0782:dT19G1, respectively. Two major DNA-binding regions (DBRs), DBR1 and DBR2, could be identified in the three complex structures, as indicated in Figure 5F and G. In the SP 0782:dT6 or SP 0782:dT12 structures, 2-4 nucleotides were bound with each subunit of the dimer, occupying both DBRs (Figure 5B-D). In contrast, DNA was present only in one subunit of the dimer in the SP 0782:dT19G1 structure, and only the DBR2 was occupied by a two-nucleotide stretch ( Figure 5E).
In the SP 0782:dT12 crystal structure, four nucleotides were bound by each SP 0782 subunit (Figures 5D and 6A). Eleven residues, E11, F12, N26, K28, W30, F39, N40, M58, K60, G61 and T63, are involved in direct or indirect ss-DNA binding ( Figure 6A), among which W30, F39, N40, M58, K60, G61 and T63 are highly conserved. In DBR1, the bases of T1 and T2, and the indole group of W30 form 'three-layer' hydrophobic stacking interactions. The T1 and T2 bases are sandwiched by the side chains of residues K28 and K60 on their edges, while the T2 base is further stabilized by residues N26/K60 and a water molecule through hydrogen bonding interactions. In DBR2, the ribose of T3 interacts with the side chain of M58, and the ribose and base of T4 pack against the side chain of F39 and F12, constituting the primary hydrophobic interaction network. The base and phosphate of T4 are further stabilized by direct or water-mediated hydrogen bonding interactions with residues E11, N40 and G61, and several polar interactions are also worth noting ( Figure 6A). In addition, a fifth nucleotide with only the phosphate group modeled participates in a hydrogen bonding network ( Figure 6A). In general, hydrophobic interactions form the core of the protein-DNA interface, with polar interactions stabilizing the peripherals for both DBRs.
In comparison with the NMR titration data, the backbone atoms of F12, F39, N40, M58, K60 and T63 showed marked CSPs (Figure 4), while the side-chain atoms of N26 and W30 also displayed marked chemical shift changes (Supplementary Figure S8), indicating a high consistency between the crystal structure and the NMR data. The backbone atom of K28 did not show any significant CSP, which may be due to the fact that the interaction of K28 with DNA is conducted by its side-chain atoms, similar to N26 and W30. However, the resonances of K28 side-chain atoms cannot be observed in the 1 H-15 N HSQC spectrum. The contacts of E11 and G61 with DNA are mediated by water molecules revealed by the crystal structure, which may be weak in the solution environment and were not detected in the NMR titration.
Except for fewer nucleotides resolved, the ssDNAprotein interaction details in the two SP 0782:dT6 structures are largely consistent with those in the SP 0782:dT12 complex, with subtle differences such as the loss of three hydrogen bonds from residues E11, K60 and T63 in the form 2 of the SP 0782:dT6 structure ( Figure 6B). Interestingly, in the SP 0782:dT19G1 structure, only two nucleotides were resolved in one subunit of the SP 0782 dimer, and the phosphate of T4 establishes a direct hydrogen bond with G61 and a salt bridge with the guanidinium group of R49, resulting in a closer contact between the sugar-phosphate backbone of the T3 and T4 nucleotides and the DBR2. Accordingly, residues 59-61 undergo a ␤-strand to loop rearrangement, resulting in a conformation similar to that in the apo NMR structure (Figures 5E and 6C). A similar local structure rearrangement was depicted previously between the apo and dT19G1-bound YdbC (16), while the PC4 and MoSub1 proteins adopt a loop structure naturally in this corresponding region (9,38) (discussed below in Figure 7). Taken together, these structures of SP 0782:ssDNA complexes reveal that the varied binding patterns and affinities of SP 0782 for different lengths of ssDNA are associated with distinct capture of nucleotides in the two DBRs of SP 0782.

The potential dimer-dimer interaction of SP 0782 in ssDNA binding
As foregoing shown, two or more SP 0782 dimers could bind with dT19G1 and form large complexes in a potential high-density manner. A model for this binding was deduced from the crystal packing of the SP 0782:dT19G1 complex (Supplementary Figure S9A). In this model, direct interaction between neighbouring SP 0782 dimers was suggested, as the ␤2/␤3-turn (residues 40-43) from the ssDNA-bound subunit of a SP 0782 dimer stacks with the W30 residue in the DBR1 from the ssDNA-bound subunit of a downstream SP 0782 dimer, while the ␣-helix of the same subunit of the upstream SP 0782 dimer stretches into the DBR2 of the ssDNA-unbound subunit of the downstream dimer ( Supplementary Figures S9A and S10A). In order to validate the dimer-dimer interaction in the deduced model, several mutants expected to weaken the interaction between SP 0782 dimers were obtained, among which the W30A mutant substituting W30 with an alanine (W30A) showed a significantly smaller size of the dT19G1-bound complex than that of the wild-type SP 0782:dT19G1 complex in EMSA (Supplementary Figure S10B). The W30A mutant existed as a homodimer, similar to wild-type SP 0782 (Supplementary Figure S11A). Thus, the smaller size of the W30A:dT19G1 complex should be due to a less number of W30A dimer binding to dT19G1. Subsequently, the stoichiometry of one subunit of the W30A dimer for dT19G1 was determined to be 0.45 by ITC (Supplementary Figure S11B), suggesting that one dimer binds with one dT19G1 strand. Moreover, only the complex containing one W30A dimer and one dT19G1 was found in the SEC-MALS assay under similar conditions for wild-type SP 0782 (Supplementary Figure S11A). These results indicated that the W30A mutation reduced the formation of large SP 0782:dT19G1 complexes containing two or more dimers, and supported the dimer-dimer interaction of SP 0782 dimers during dT19G1 binding. In addition, the W30A mutant displayed significantly decreased affinity for dT19G1 (4.12 × 10 −5 M) in ITC and EMSA experiments (Supplementary Figures S10B and S11B), confirming the important role of W30 in nucleotide binding, as revealed by the crystal structures of the SP 0782:ssDNA complexes,.

DISCUSSION
PC4-like proteins have OB fold-derived structures and form homodimers to bind with ssDNA, and the representative protein, human PC4, was originally identified as a transcriptional coactivator playing a role during transcription (9,11). Subsequent studies have revealed broad roles of PC4 and Sub1, its close homolog from yeast, in chromatin condensation, maintenance of genome stability and DNA repair (3). Moreover, several PC4-like proteins in bacteria have been identified, including YdbC in L. lactis and BPSL1147 in B. pseudomallei (16,18). However, the structure and function of the bacteria PC4-like proteins are still largely unknown. In this study, we identify and characterize SP 0782, a novel PC4-like protein in S. pneumoniae that has an ssDNA-binding property and mode different from those of PC4 and YdbC, which will facilitate the understanding of the DNA-binding mechanism of PC4-like proteins.

The variation in ssDNA length bound by PC4-like proteins
Human PC4 shows no significant binding for ssDNA shorter than dT10, while 16 or more nucleotides are needed to reach the affinity of nano-molar level (10). If the length of ssDNA extends to 28 nt, two dimers of PC4 would be steadily bound (12,13). These binding properties of human PC4 are consistent with its crystal structure bound with dT19G1, which shows that each subunit of the PC4 dimer binds with five nucleotides (9). YdbC shows an affinity of nano-molar level for dT19G1, whereas two dimers can bind to one dT19G1 with the second dimer showing very weak and micro-molar affinity, which could be explained by its complex structure, showing that each subunit of the YdbC dimer binds with seven nucleotides (16). However, SP 0782 could bind with dT19G1 steadily in a two-dimer bound form and with dT6 in a micro-molar affinity, and could even bind with dT12 in a two-dimer bound form at a high protein:DNA ratio, suggesting that the nucleotides bound by each subunit of the SP 0782 dimer are less than those bound by PC4 and YdbC. The crystal structure of the SP 0782:dT12 complex fully supports this suggestion, wherein four nucleotides bind to the surface of each subunit of the SP 0782 dimer. Thus, it appears that PC4-like proteins are divergent in the ssDNA length of binding.

The variation in DNA-binding details in PC4-like proteins
The detailed structural differences and diverse DNA binding modes between SP 0782 and two representative PC4like proteins are summarized in Figure 7. Although the overall folds of SP 0782, YdbC and PC4 are similar, the protein:ssDNA binding details are different at the DBR1 and DBR2 regions. In the SP 0782:dT6 and SP 0782:dT12 structures, the T2 base stacks on top of the W30 side chain ( Figure 7D, left panel), and the corresponding base inserts between the W23 side chain and the G52 backbone in the YdbC:dT19G1 structure ( Figure 7D, central panel), or stacks on top of the W89 side chain in the PC4:dT19G1 structure ( Figure 7D, right panel). The W30 in SP 0782 and the W23 in YdbC are located at the beginning of the ␤2 strand, while the W89 in PC4 and the Y74 in MoSub1 (a W89 equivalent in PC4) are located at the end of the ␤3 strand. Moreover, the ␤-turns between ␤1 and ␤2 of PC4 and MoSub1 are shorter than those of SP 0782 and YdbC ( Figure 7E). Another significant difference among the PC4like family is the DBR2 region, denoted as 'closed' or 'open' form herein. Different from the naturally open DBR2 of PC4, which could readily intake two adjacent nucleotides between P98 and F77, the closed DBR2 of YdbC switched to open state by the inducement of a single-strand DNA ( Figure 7B and C). The DBR2 of SP 0782 is also closed; however, it could bind two nucleotides of various lengths of ssDNAs without large-scale structural rearrangement (Figures 6 and 7A). Thus, it can be concluded that although these PC4-like proteins adopt a conservative overall fold to build the DBRs and utilize similar hydrophobic functional groups for DNA binding, the structural details in each PC4like protein and the structural rearrangement upon ssDNA binding do vary to some extent. Importantly, two major differences between prokaryotic and eukaryotic PC4-like proteins can be summarized as the location of the key Trp residue in DBR1 and the open/closed state of DBR2 without DNA binding.

The role of DBR1 for ssDNA binding
Among the four resolved SP 0782:ssDNA complex structures, one nucleotide was resolved stacking with residue W30 in the SP 0782:dTCTTCC complex structure, two or three nucleotides were resolved occupying DBR1 and partial DBR2 regions in SP 0782:dT6 complex structures (form 1 and form 2), and four intact nucleotides were resolved occupying both DBR1 and DBR2 in SP 0782:dT12 complex structures ( Figure Figure S12), only one nucleotide was resolved stacking with W30 in the structure of the SP 0782:dTCTTCC complex, while two to four nucleotides were modeled stretching from DBR1 to DBR2 in the SP 0782:dT6 and SP 0782:dT12 structures, indicating that DBR1 may serve as an initial ssDNA binding site and DBR2 enhances the ssDNA-SP 0782 binding affinity, subsequently. Even though the overall T2-DBR1 interaction network is shared by the four resolved types of complex structures, the direct distances between T2 and K60 reduced along with the extension of modeled nucleotides, resulting in a transition from water-mediated to direct T2 and K60 interactions (Supplementary Figure S7). The observation of T2 swaying suggests that T2-DBR1 interactions may play key roles in both initializing and accommodating the binding of ssDNA at various lengths.

A potential high-density binding pattern of SP 0782
It is known that PC4 can bind with ssDNA in a twodimer-bound form (12,13). Furthermore, the crystal packing of the PC4:dT19G1 complex reveals a probable mode of DNA-mediated multimerization of the PC4 dimer, which may be relevant for the unwinding of duplex DNA by PC4 (9). Similarly, in the crystal structure of the Mo-Sub1:dT19G1 complex, two ssDNA strands mediate the multimerization of three MoSub1 dimers (Supplementary Figure S9B) (38). In this study, the formation of large complexes of SP 0782 with ssDNA was found in EMSA and SEC-MALS experiments, and a model of the DNAmediated multimerization of the SP 0782 dimer was deduced from the crystal packing of the SP 0782:dT19G1 complex (Supplementary Figure S9A). Thus, the DNAmediated multimerization seems to be a general mechanism for PC4-like proteins, and can be reasonably linked to their functions when the binding of long ssDNA is required.
In the deduced multimerization model from the SP 0782:dT19G1 structure, the distance between the 5 -phosphate of T3 and the 3 -OH of T4 from an adjacent SP 0782:dT19G1 dimer is ∼7Å, implying a disordered nucleotide (T2) between the two SP 0782 dimers (Supplementary Figure S9A). Only one subunit of each SP 0782 dimer is occupied by DNA, and each SP 0782 dimer binds with three nucleotides in the DNA strand, displaying a high-density binding. During SP 0782 and dT19G1 binding, the interaction between SP 0782 dimers facilitates the multimerization and high-density binding, suggesting a cooperative mechanism of SP 0782 dimers. Nevertheless, the multimerization models for PC4, MoSub1 and SP 0782 revealed by the crystal packing of the protein:dT19G1 complexes are different to some extent. Whether the conserved Trp residue in DBR1 is also essential for the multimerization of PC4 and MoSub1 requires further investigation.

The potential subunit-occupied modes of SP 0782
The high-density binding model of SP 0782 implies a potential single-subunit-occupied (SSO) mode, wherein only one subunit of the SP 0782 dimer is occupied by DNA, in contrast to the double-subunit-occupied (DSO) mode revealed by the complex structures of SP 0782:dT6 and SP 0782:dT12. The length-dependent DNA-binding patterns of SP 0782 can be summarized and classified into the SSO and DSO modes, respectively ( Supplementary Figure S13). One SP 0782 dimer can bind with one (SSO) or two dT6 oligos (DSO) in a micro-molar affinity, while one SP 0782 dimer bind with one dT12 (DSO) in a nano-molar affinity likely achieved by a cooperation between the two subunits in the same dimer. Moreover, two SP 0782 dimers may bind with one dT12 (SSO) at high SP 0782:ssDNA molar ratios in a potential high-density manner, similar to the binding of dT19G1 by SP 0782 (Supplementary Figure  S9A). The situation for dT19G1 is more complicated. Besides the binding pattern containing two SP 0782 dimers and one dT19G1 (a hybrid of SSO and DSO) detected in ITC and SEC-MALS experiments, other patterns, including one SP 0782 dimer versus one dT19G1 (DSO), three SP 0782 dimers versus two dT19G1 oligos (DSO), and three SP 0782 dimers versus one dT19G1 (SSO), could also be adopted.
The SSO and DSO modes may undergo transitions when the SP 0782:ssDNA ratio changes (Supplementary Figure  S13), as implied by the EMSA experiments. This is reminiscent of the transition of EcSSB between the (SSB) 35 and (SSB) 65 modes during ssDNA binding. EcSSB binds 35 nt ssDNA with two subunits of its tetramer ((SSB) 35 mode) and 65 nt ssDNA with all four subunits of its tetramer ((SSB) 65 mode), but at a high protein:DNA ratio, EcoSSB could also bind with 65 nt ssDNA with two tetramers bound in (SSB) 35 mode to achieve high-density binding (reviewed in (39)). Only two subunits of each Ec-SSB tetramer bind with DNA, and EcSSB tetramers interact with each other to achieve high-density binding in a cooperative manner (reviewed in (39)). Therein, the unoccupied DNA-binding sites in one tetramer interact with the C-terminal conserved nine-amino acid tip (TIP) from a neighboring tetramer (40), analogous to the direct contacts between neighbouring SP 0782 dimers deduced in the highdensity binding model (Supplementary Figure S9A). Nevertheless, the hypothesis of SSO mode and the transition between SSO and DSO modes of SP 0782 demands further demonstration in future.

The biological function of SP 0782
Broad roles of PC4 and Sub1 in transcription and maintenance of genome stability have been implicated. The role of PC4 in maintenance of genome stability depends on its CTD (41). In yeast and in the context of ectopic expression in E. coli, eukaryotic PC4 could protect chromosomal DNA against oxidative damage via a mechanism that strictly depends on the ssDNA-binding domain (42,43). Thus, it is expected that SP 0782 may be involved in DNA protection and repair under oxidative stress in S. pneumoniae, which are important for pathogenic bacteria survival, especially during the infection process, because host immune cells usually produce an oxidative burst aimed to kill the pathogens. Meanwhile, it is worth noting that SP 0782 may be involved in natural transformation in S. pneumoniae. Natural transformation is a biological process in which bacteria intake exogenous DNA and incorporate it into their genomes by recombination, which facilitates genome evolution and fitness (44,45). SpSsbB has been shown to be involved in the natural transformation of S. pneumoniae through binding exogenous ssDNA, and may facilitate the ssDNA binding by SpDprA and then loading to recombination-related protein RecA (46)(47)(48)(49). A pioneering study showed that the expression level of the SP 0782 gene was up-regulated eight-fold during competence induction, which is a status of S. pneumoniae cells for natural transformation (50). However, the deletion of the SP 0782 gene did not significantly change the competence induction of S. pneumoniae cells. Considering that the ssDNA binding function of SP 0782 was not revealed until our study, more well-designed experiments may be required to figure out whether SP 0782 is associated with the natural transformation of S. pneumoniae.

CONCLUSION
In summary, SP 0782 represents a novel PC4-like protein that binds with ssDNA in S. pneumoniae. The SP 0782 dimer has a conserved PC4-like fold, and binds to two antiparallel ssDNA strands with each subunit. The two subunits in one SP 0782 dimer bind with ssDNA cooperatively to achieve a high affinity. The ssDNA length fully occupied by SP 0782 is shorter than those occupied by PC4 and YdbC, which is associated with the different structures of the ssDNA-binding interface. SP 0782 exhibits varied binding patterns for different lengths of ssDNA, which result from distinct capture of nucleotides in the two major DNA-binding regions in SP 0782. Furthermore, SP 0782 tends to form a large complex with ssDNA at a high SP 0782:ssDNA molar ratio, and a high-density binding model involving cooperation between SP 0782 dimers during ssDNA binding is proposed. According to the binding patterns of SP 0782 dependent on DNA length, two different subunit-occupied modes of SSO and DSO are suggested, along with a hypothesis of the transition of SP 0782 between the SSO and DSO modes due to the change in SP 0782:ssDNA molar ratio. In addition, the biological roles of SP 0782, probably in DNA protection and repair, as Nucleic Acids Research, 2020, Vol. 48, No. 1 443 well as natural transformation are discussed. Our study provides insights into the DNA-binding mechanism of SP 0782 and even PC4-like proteins, and benefits further studies regarding the DNA-binding dynamics and the biological function of SP 0782.

DATA AVAILABILITY
The atomic coordinates and NMR restraints for the reported NMR structure have been deposited at the Protein Data Bank under accession number 2L3A. The atomic coordinates and structure factors for the reported crystal structures have been deposited at the Protein Data bank under accession numbers 5ZKL, 5ZKM, 6JIP, and 6JIQ.