title: Bacterial expression of the HINab domain of IFI16: purification, characterization of DNA binding activity, and co-crystallization with viral dsDNA.
abstract: Interferon-inducible protein 16 (IFI16) is a multifunctional p200-family protein that plays pivotal roles in p53-mediated apoptosis, tumor suppression and DNA damage response. Recently, another function of IFI16 in innate immune sensing and response has been uncovered, in which IFI16 recognizes the exogenous DNAs through cooperative binding of DNAs via its two DNA binding domains, HINa and HINb. Although the mechanism by which the HINb domain recognizes DNAs has been elucidated, the molecular basis of the cooperativity between HINa and HINb during DNA recognition process is still not clear. Here we report expression and purification of a truncated human IFI16 protein (HINab-GS) containing HINa in tandem with HINb with the joining region between HINa and HINb replaced by a short GS linker in Escherichia coli. DNA binding activities of HINab-GS to various double-stranded DNAs (dsDNAs) of different lengths were then examined using electrophoretic mobility shift assays. HINab-GS exhibited efficient binding activity to dsDNAs, and its DNA binding affinity correlated positively with the length of dsDNAs. A co-crystallization condition of HINab-GS bound to a 30 bp dsDNA derived from vaccinia virus was also found. Together, our work provides a basis for structurally elucidating the mechanism governing cooperative DNA recognition by IFI16.
authors: Life Sciences Institute, Zhejiang University, Hangzhou 310058, PR China; Department of Pharmacy, Xiamen Medical College, Xiamen 361008, PR China. Electronic address: zhouliu@zju.edu.cn.
authors: Zhou
authors: Z
authors: Liu
authors: Life Sciences Institute, Zhejiang University, Hangzhou 310058, PR China.
authors: Xiaoxiao
authors: X
authors: Zheng
authors: Life Sciences Institute, Zhejiang University, Hangzhou 310058, PR China.
authors: Yiwen
authors: Y
authors: Wang
authors: Life Sciences Institute, Zhejiang University, Hangzhou 310058, PR China. Electronic address: haiwei@zju.edu.cn.
authors: Haiwei
authors: H
authors: Song
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004279:DNA, Viral / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004279:DNA, Viral; D009687:Nuclear Proteins; D010750:Phosphoproteins; C076700:IFI16 protein, human
keywords: Crystallization; DNA binding activity; Exogenous DNA sensor; Innate immune response; Interferon-inducible protein 16; Vaccinia virus