New insights into the structural basis of DNA recognition by HINa and HINb domains of IFI16

doi:10.1093/jmcb/mjv053 Published online August 5, 2015 Journal of Molecular Cell Biology (2016), 8(1), 51 –61 | 51 Article New insights into the structural basis of DNA recognition by HINa and HINb domains of IFI16 Xiangmin Ni1,2,† , Heng Ru2,†, Feng Ma3,† , Lixia Zhao2,†, Neil Shaw2, Yingang Feng4, Wei Ding2, Weibin Gong2, Qiaofeng Wang1, Songying Ouyang2,*, Genhong Cheng3,*, and Zhi-Jie Liu1,2,5,* 1 Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 3 Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA 4 The Qingdao Engineering Laboratory of Single Cell Oil and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China 5 iHuman Institute, ShanghaiTech University, Shanghai 201210, China † These authors contributed equally to this work. * Correspondence to: Zhi-Jie Liu, E-mail: ; Genhong Cheng, E-mail: ; Songying Ouyang, E-mail: 2 Interferon gamma-inducible protein 16 (IFI16) senses DNA in the cytoplasm and the nucleus by using two tandem hematopoietic interferon-inducible nuclear (HIN) domains, HINa and HINb, through the cooperative assembly of IFI16 filaments on double-stranded DNA (dsDNA). The role of HINa in sensing DNA is not clearly understood. Here, we describe the crystal structure of the HINa domain in complex with DNA at 2.55 Å resolution and provide the first insight into the mode of DNA binding by the HINa domain. The structure reveals the presence of two oligosaccharide/nucleotide-binding (OB) folds with a unique DNA-binding surface. HINa uses loop L45 of the canonical OB2 fold to bind to the DNA backbone. The dsDNA is recognized as two single strands of DNA. Interestingly, deletion of HINb compromises the ability of IFI16 to induce IFN-b, while HINa mutants impaired in DNA binding enhance the production of IFN-b. These results shed light on the roles of IFI16 HIN domains in DNA recognition and innate immune responses. Keywords: interferon gamma-inducible protein 16 (IFI16), hematopoietic interferon-inducible nuclear (HIN) domain, DNA recognition, innate immune responses Introduction Host cells possess intricate innate immune machinery for the detection of microbial DNA and RNA. Upon sensing the conserved pathogen-associated molecular patterns (PAMPs) associated with nucleic acids, a cascade of signaling steps is activated, culminating in the production of type I interferons (IFNs) and pro-inflammatory cytokines (Ishii and Akira, 2006; Muruve et al., 2008; Hornung and Latz, 2010). A large number of DNA sensors operating in various cell types have been identified and characterized. These sensors include TLR9 functioning in the endosomes of plasmacytoid dendritic cells (Blasius and Beutler, 2010; Kawai and Akira, 2010), DAI in mouse embryonic fibroblasts (Takaoka et al., 2007), RNA polymerase III in BMDMs, HEK, and other cell types (Ablasser et al., 2009; Chiu et al., 2009), and LRRFIP1 in primary peritoneal macrophages (Yang et al., 2010). In addition to these distinct DNA sensors that function in different cell types, several cytosolic DNA sensors share DNA-binding domains and belong to a family. For example, the DExD/H-box domain-containing family of helicases Received November 27, 2014. Revised April 24, 2015. Accepted April 30, 2015. # The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. includes members such as DHX36 that recognizes the CpG A oligodeoxynucleotide (ODN) (Kim et al., 2010), DHX9 that detects the CpG B ODN (Zhang et al., 2011c), DDX1 and DHX9 that sense poly I:C (Zhang et al., 2011a), and DDX41 that senses doublestranded DNA (dsDNA) (Zhang et al., 2011b). Recently, Sun et al. (2013) identified cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) as a cytosolic DNA sensor. The recognition of cytoplasmic DNA by cGAS activates the enzyme to catalyze the noncanonical cyclic dinucleotide from GTP and ATP (Diner et al., 2013; Gao et al., 2013a; Zhang et al., 2013). Subsequently, cGAMP activates the receptor STING to recruit TBK1, which then activates the transcription factors IRF3 and NF-kB, resulting in the production of type I interferon and other cytokines (Gao et al., 2013b; Zhang et al., 2013). In contrast, members belonging to the second family of cytosolic DNA sensors, the PYHIN family, contain a pyrin (PYD) and one or more hematopoietic interferon-inducible nuclear (HIN) protein domains containing 200 amino acids (Schattgen and Fitzgerald, 2011). Thus far, four proteins—absent in melanoma 2 (AIM2), interferon gamma-inducible protein 16 (IFI16), interferon-inducible protein X (IFIX), and myeloid cell nuclear differentiation antigen (MNDA)—in humans and 13 proteins in mice have been shown to 52 | Ni et al. be members of this family (Brunette et al., 2012). Together, these proteins are called AIM2-like receptors (ALRs) (Unterholzner et al., 2010). Except for murine p202, all these receptors contain a PYD. P202 contains two HIN domains, HINa and HINb, but lacks a PYD. The innate immune responses originating from AIM2 are exerted via the formation of a large complex called the inflammasome (Burckstummer et al., 2009; Fernandes-Alnemri et al., 2009; Hornung et al., 2009). Upon the binding of DNA by the HIN domain, the PYD recruits the adaptor protein ASC via PYD–PYD interactions. ASC in turn recruits pro-caspase 1 through CARD–CARD interactions. Multiple molecules of IFI16 bound to DNA ensure filament formation as a broad host defense strategy (Morrone et al., 2014), bringing caspases into proximity for inter-molecular proteolytic activation. Activated caspase 1 processes IL-1b and IL-18 into mature pro-inflammatory forms, ultimately leading to pyroptosis. P202 has been shown to function as an antagonist of AIM2 function by sequestering DNA or heterodimerizing with AIM2 and preventing molecular crowding (Roberts et al., 2009). This activity results in the termination of AIM2-mediated responses. Among the known DNA sensors, thus far, only IFI16 has been shown to be capable of sensing DNA in both the nucleus and the cytoplasm and of activating innate immune responses (Unterholzner et al., 2010; Kerur et al., 2011). These IFI16 functions are executed via a domain architecture consisting of an N-terminal PYD followed by two tandem HIN domains: HINa and HINb (Figure 1A). Whereas the sensing of Kaposi Sarcoma-associated herpes virus (KSHV) dsDNA in the nucleus by IFI16 results in the assembly of an AIM2-independent inflammasome and maturation of IL-1b and IL-18, the detection of dsDNA by IFI16 in the cytoplasm leads to the activation of the STING–TBK1– IRF3 pathway and the produc (...truncated)