E coli SSB and its binding modes 74

To date, E. coli SSB is the best-characterised prokaryotic SSB protein. It functions as a

homotetramer. Each subunit of E. coli SSB consists of two domains. The N-terminal

domain (residues 1-112) is composed of an oligonucleotide/oligosaccharide binding fold (OB fold), which binds to ssDNA (Lohman and Ferrari, 1994). Chemical modification studies and fluorescence quenching experiments suggested that tryptophan (Trp) residues are important for ssDNA binding (Overman et al., 1988; Curth et al., 1993). Trp-54 and Trp-88 were shown to play a major role in ssDNA binding as nearly complete fluorescence quenching was observed for these residues (Curth et al, 1993). Furthermore, Trp-40 and Trp-54 were shown to form stacking interactions with ssDNA bases (Khamis et al., 1987). In addition, crosslinking experiments (Merrill et al., 1984)

and mutational studies (Casas-Finet, 1987; Bayer, 1989) indicated that Phe-60 is involved in ssDNA binding while also affecting the stability of the tetramer.

Figure 3.2 X-ray crystal structure of the E. coli SSB tetramer (PDB ID: 1SRU; Savvides et al., 2004).

The C-terminal domain of SSB (residues 113-177) consists of a long intrinsically disordered and flexible polypeptide chain with eight predominantly negatively charged residues (DFDDDIPF) at the C-terminal tip (Shereda et al., 2008). These eight residues are highly conserved among bacterial SSBs (Lu and Keck, 2008). They bind to the OB domain (Shishmarev et al., 2014) and, by binding to SIPs, they assist with recruiting SIPs to ssDNA (Shereda et al., 2008). The NMR experiments showed that the C-terminal tip is in fast exchange between unbound and bound states to the OB domain. Furthermore, the C-terminal tip is mostly in the unbound state even in the absence of ssDNA (Su et al., 2014).

Due to the presence of four OB domains in the tetramer, SSB can bind long ssDNA strands in multiple binding modes, which are characterised by the length of occluded ssDNA segments. This has been observed by electron microscopy, where Griffith et al. (1984) observed different ssDNA binding modes when varying the protein to DNA ratio, as well as in solution, where Lohman and Overman (1985) reported

different ssDNA binding modes at different salt conditions, which resulted in different quenching behaviour of the intrinsic SSB fluorescence.

Among the different binding modes reported, the (SSB)35 and (SSB)65 modes are

the two major binding modes observed in vitro (Lohman and Overman, 1985). The

relative abundance of these two binding modes depends on the solution conditions. The valance of any salt and its concentration, as well as the ratio of protein to DNA are the two main factors, which determine the relative abundance and stabilities of these two binding modes. In addition, there are effects from the pH and temperature (Lohman and Overman, 1985; Bujalowski and Lohman, 1986; Bujalowski et al., 1988; Wei et al., 1992).

Figure 3.3 E. coli SSB binding modes. (A) Schematic representation of the SSB-

ssDNA interaction in the (SSB)65 binding mode, with 65 nucleotides of DNA (orange

ribbon) wrapped around each SSB tetramer. (B) Schematic drawing of a hypothetical

model of the SSB-ssDNA complex in the (SSB)35 binding mode, where pairs of SSB

tetramers interact with ssDNA segments of about 70 nucleotides (orange line) using an average of only two subunits of each tetramer. Reproduced from Kozlov et al. (2017).

The (SSB)35 mode is favoured at low monovalent salt concentrations ([NaCl] <

10 mM) and high ratios of protein to DNA. In this mode ssDNA wraps around, on average, only two of the four subunits in the SSB tetramer. For long segments of ssDNA, SSB can bind with very high cooperativity, forming long protein clusters (Figure 3.3B; Griffith et al., 1984; Lohman et al., 1986; Ferrari et al., 1994). On the

other hand, the (SSB)65 mode is favoured at moderate to high monovalent salt

concentrations ([NaCl] > 200 mM). In this mode, ssDNA can occupy all four possible

binding sites on SSB (Figure 3.3A). Unlike the (SSB)35 mode, the (SSB)65 mode

displays limited cooperativity between tetramers, forming at most dimers of tetramers (octamers; Chrysogelos and Griffith, 1982; Griffith et al., 1984; Bujalowski and Lohman, 1987; Overman et al., 1988).

Since ssDNA binds to the four subunits of SSB with salt-dependent negative cooperativity, the relative stability of the two main binding modes can be modulated by the monovalent salt concentration in solution. The negative cooperativity effect is strong at low salt and decreases with increasing salt concentration. At low salt concentration, ssDNA can bind tightly to the first two subunits but binding to the other two subunits is much weaker (Lohman and Bujalowski, 1988; Bujalowski and Lohman, 1986). Salt dependence of the negative cooperativity effect is the main reason for the transition between the two major binding modes with changing salt concentrations.

A crystal structure of a chymotryptic fragment of the SSB tetramer bound to two

molecules of dC35 has been published, where the chymotryptic digestion served to

remove the unstructured C-terminal polypeptide segment of SSB (Figure 3.4; Raghunathan et al., 2000). Based on this structure, models have been proposed for the

topologies of ssDNA wrapping around SSB in the (SSB)65 and (SSB)35 modes. In the

model proposed for the (SSB)65 mode, the ssDNA wraps around all four subunits of

SSB with a topology similar to a baseball seam. In this model, the 5’ and 3’ ends of the ssDNA enter and exit the tetramer in close proximity (Raghunathan et al., 2000). The

model proposed for the (SSB)35 mode shows partial interactions with three monomer

units, which on average equal interactions with two subunits (Figure 3.3B). In this model, the ssDNA enters and exits the tetramer on opposite sides (Raghunathan et al., 2000). Notably, the crystal structure did not detect electron density for all nucleotides, making these models to a large extent speculative.

Figure 3.4 X-ray crystal structure of a chymotryptic fragment of E. coli SSB bound to two 35-mer single-stranded DNA molecules (PDB ID: 1EYG; Raghunathan et al., 2000).