Intrinsically disordered C-terminal tails of E. coli single-stranded DNA binding protein regulate cooperative binding to single-stranded DNA

Abstract

The homotetrameric Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA replication, repair and recombination. E. coli SSB can bind to long single-stranded DNA (ssDNA) in multiple binding modes using all four subunits [(SSB)65 mode] or only two subunits [(SSB)35 binding mode], with the binding mode preference regulated by salt concentration and SSB binding density. These binding modes display very different ssDNA binding properties with the (SSB)35 mode displaying highly cooperative binding to ssDNA. SSB tetramers also bind an array of partner proteins, recruiting them to their sites of action. This is achieved through interactions with the last 9 amino acids (acidic tip) of the intrinsically disordered linkers (IDLs) within the four C-terminal tails connected to the ssDNA binding domains. Here, we show that the amino acid composition and length of the IDL affects the ssDNA binding mode preferences of SSB protein. Surprisingly, the number of IDLs and the lengths of individual IDLs together with the acidic tip contribute to highly cooperative binding in the (SSB)35 binding mode. Hydrodynamic studies and atomistic simulations suggest that the E. coli SSB IDLs show a preference for forming an ensemble of globular conformations, whereas the IDL from Plasmodium falciparum SSB forms an ensemble of more extended random coils. The more globular conformations correlate with cooperative binding.

Keywords: DNA repair; DNA replication; cooperativity; regulation; simulations.

Figure 1. Structural organization of E. coli SSB linker variants

(a) wt EcSSB subunits (177 aa) are comprised of an N-terminal DNA binding domain (OB-fold) (1-112 residues), and a C-terminal intrinsically disordered (ID) linker (56 aa) and a nine-residue acidic “tip”. (b) Structural model of 65 nucleotides of ssDNA (red ribbon) bound to the EcSSB tetramer [15] with the addition of the C-terminal tails (shown in grey) that are not observed in the crystal structure. (c) Design of SSB-linker variants containing the Ec core (1-112 aa), but varying C-terminal tails: missing part of the linker (i), the whole linker (GG, (ii)), acidic “tip” (SSBΔC8, (iii)) or replacing the Ec linker with the Pf. linker (EcPfEc, (iv)).

Figure 2. Results from simulations summarizing the conformational properties of IDLs

(a) Plot of the internal scaling profiles for the four EcEc linker tail variants and the PfPf linker + tail. The profiles for globular (GR), Flory random coils (FRC), and self-avoiding reference (SAR) ensembles are also shown for calibration. (b) Plot of the cumulative distribution functions (CDFs) of R_ee values for each of the linker variants. The mean R_ee values are 22.6 ± 0.7Å for EcEc, 17.8 ± 0.8Å for EcEc(Δ151-166), 16.8 ± 1.9Å for EcEc(Δ130-166), 13.7 ± 0.1Å for EcEc(Δ120-166) and 63.6 ± 0.9 Å for PfPf.

Figure 3. Occluded site sizes of SSB linker variants on poly(dT)

Results of titrations of 0.30 μM of wtSSB (cyan), SSB-GG (orange) and SSB-EcPfEc (dark grey) with poly(dT) monitoring SSB Trp fluorescence quenching in buffer T, 25°C with (a) 0.30 M NaCl or (b) 10mM NaCl to estimate the occluded site sizes (n) for the SSB variants bound to poly(dT) [54].

Figure 4. Cooperativity of SSB linker variants bound to M13 ssDNA

(a) - EMSAs of SSB-M13mp18 ssDNA complexes in buffer T, 10mM NaCl, 22°, formed at different protein/DNA ratios: R₃₅=35[SSB]_tot/[M13nts]_tot. wtSSB (a-i) shows a bimodal distribution of bound DNA at intermediate ratios (R₃₅=0.05–0.7) indicative of highly cooperative binding, whereas SSB-GG (a-ii) and SSB-EcPfEc (a-iii) show single band at all protein:DNA ratios indicative of low or no cooperativity. (b) - Sedimentation velocity profiles of SSB variants bound to M13 ssDNA at R₃₅=0.3 (buffer T, 10mM NaCl, 25°C). Bimodal distribution of wtSSB-M13 complexes (b-i) indicates highly cooperative binding. SSB-GG and SSB-EcPfEc (b-ii) bind with low cooperativity. Intermediate linker deletion constructs SSBΔ130-166 and SSBΔ120-166, as well as two-tail variant LD-Drl retain high cooperativity (b-iii). SSB missing conserved acidic “tip” (SSBΔC8) and single tail SSB construct, LT-Drl, (b-iv) show decreased cooperativity (see Table S1 for quantification).

Fig. 5. Sensitivity of E. coli strains carrying the SSB linker variants to UV irradiation

The sensitivity to uv irradiation of E. coli cells carrying the SSB linker deletion variants SSBΔ151-166, SSBΔ130-166 and SSBΔ120-166 is very similar to wtSSB, although sensitivity increases for the SSB-EcPfEc variant and even more for the SSB-GG variant.

Figure 6. Model for highly cooperative ssDNA binding in the (SSB)₃₅ mode

Binding of SSB to ssDNA in its (SSB)₃₅ binding mode leaves DNA binding sites unoccupied in two subunits. Negatively charged tips of two C-terminal tails bind to the unoccupied subunits of adjacent tetramers. Positive cooperativity is enhanced due to inter-chain interactions between globular IDLs of adjacent tetramers.