Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction

Abstract

The bacterial single stranded DNA binding protein SSB is a strictly conserved and essential protein involved in diverse functions of DNA metabolism, including replication and repair. SSB comprises a wellcharacterised tetrameric core of N-terminal oligonucleotide binding (OB) folds that bind single-stranded DNA (ssDNA) and four intrinsically disordered C-terminal domains of unknown structure that interact with partner proteins. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. In this structural study, we show that the opposite is true. Combined small angle scattering with X-rays and neutrons coupled to coarse-grained modelling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered Cterminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

“…The IDL is rich in Gly (17), Pro (9) and Gln plus Asn (14). As a result of this composition, the E. coli IDL is predicted to form an ensemble of compact globular conformations [27], consistent with hydrodynamic studies [27] and small angle x-ray and neutron scattering studies [56]. Such globular conformations may promote positive cooperativity through linker-linker interactions upon ssDNA binding.…”

The IDL of E. coli SSB links ssDNA and protein binding by mediating protein–protein interactions

“…This sequence composition likely contributes to the movements of the C-terminal domain of the protein associated with ssDNA binding. 45 Our analysis of the linker region of EcSSB suggests that the amino acids present in the region from Q117 to Q147 have a propensity to form a PPII-like helix. [46][47][48] This is supported by modeling which showed that this region could adopt a structure resembling a collagen monomer.…”

“…The presence of this collection of repeats confers elastomeric properties such as those observed in the eukaryotic proteins enabling the C‐terminal 69 residues to adopt different conformations thereby facilitating a larger range of partner proteins with which SSB can bind. This sequence composition likely contributes to the movements of the C‐terminal domain of the protein associated with ssDNA binding …”

Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

“…While phosphorylation of the tyrosine 82 dramatically increased the SsbA affinity for single-stranded DNA (6), phosphorylation of the threonine 38 enhanced the capacity of SsbA tetramers to attach adjacently to a single-stranded DNA substrate. It was recently demonstrated that the disordered C-terminal region of bacterial Ssb proteins collapses towards the tetramer upon DNA-binding (20), thus leading to a more compact structure. It is tempting to speculate that phosphorylation of threonine 38 could lead to a specific interaction with the C-terminal domains of adjacent SsbA monomers, and thereby aid the compacting process upon binding single-stranded DNA.…”