Nigam Richa, Mohan Monisha, Shivange Gururaj, Dewangan Pranjal Kumar, Anindya Roy
Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, Telangana, 502285, India.
Mol Biol Rep. 2018 Oct;45(5):865-870. doi: 10.1007/s11033-018-4232-6. Epub 2018 Jul 4.
Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152-169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.
蛋白质的内在无序区域（IDR）通常通过与折叠结构域的相互作用来调节功能。大肠杆菌单链DNA结合蛋白SSB能结合并稳定单链DNA（ssDNA）。SSB的N端含有特征性的OB（寡核苷酸/寡糖结合）折叠结构，可紧密但非特异性地结合ssDNA。SSB还通过主要由酸性氨基酸残基组成的C端相互作用结构域与大量蛋白质形成复合物。已知位于OB折叠结构和C端酸性结构域之间的氨基酸残基构成一个IDR，且该区域尚无功能意义。尽管已知SSB能结合许多DNA修复蛋白，但尚不清楚它是否能与DNA脱烷基化修复蛋白AlkB结合。在此，我们对AlkB与SSB的相互作用进行了表征，并证明SSB通过IDR与AlkB结合。我们通过体外下拉实验和酵母双杂交分析确定了AlkB与SSB的相互作用。我们将相互作用位点定位到SSB的152 - 169位残基。与大多数利用C端酸性结构域进行相互作用的SSB结合蛋白不同，SSB的IDR对于与AlkB的相互作用是必要且充分的。
