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

ABSTRACT: The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.

SUBMITTER: Bianco PR

PROVIDER: S-EPMC5275737 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Bianco Piero R PR Pottinger Sasheen S Tan Hui Yin HY Nguyenduc Trong T Rex Kervin K Varshney Umesh U

Protein science : a publication of the Protein Society 20170126 2

The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When inter ...[more]

PMID: 28127816

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Project description:Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB)35) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (?35), forming long protein clusters, whereas the 65-site-size ((SSB)65) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays "limited" cooperativity (?65), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB)65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearest-neighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT)70 and (dT)140. We find that the (SSB)35 mode always shows extremely high "unlimited" cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-?L, bind in the (SSB)65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (?65/35), with ?65 ? ?65/35 <?35. The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearest-neighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity.

Dataset Information

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

Publications

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

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