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DEAD-box proteins, which are named after the strictly conserved amino acid sequence Asp-Glu-Ala-Asp, were first identified as a distinct family in the late 1980s when alignments based on eight homologues of the yeast eIF4A highlighted the presence of several conserved motifs (Linder et al., 1989). DEAD-box proteins are widely distributed in different life forms, ranging from bacteria to human and constitute the largest RNA helicase family (Jiang et al., 2016). They are involved in many aspects of RNA metabolism, such as splicing, mRNA export, transcriptional and translational regulation, ribosome biogenesis and RNA decay (Rocak and Linder, 2004). The core of DEAD-box proteins is organized into two major domains. Domain 1 (DEAD domain) consists of motifs Q, I (Walker A, P-loop), II (Walker B, DEAD-box), Ia, GG, Ib and III, whereas domain 2 (Helicase domain) consists of motifs IV, V and VI. Different motifs are involved in nucleotide binding (Q, I and II), RNA binding (Ia, Ib, IV and V) and ATP hydrolysis (III and possibly VI). Compared with the two conserved domains, the Nand C-terminal regions are variable and divergent. Their functions are not fully characterized, but they are thought to confer their own specificity on different proteins (Hogbom et al., 2007). The recognition of pathogen-associated molecular patterns (PAMPs) of pathogens by pattern recognition receptors (PRRs) is important for the induction of type I interferons (IFN) (Medzhitov and Janeway, 2000). DDX41, a member of the DEAD-box proteins, containing a disordered N-terminal region, a DEAD domain and a Helicase domain (Fig. 1A), was identified as an intracellular DNA sensor in myeloid dendritic cells (mDCs) by Yong-Jun Liu’s group. They showed that DDX41 directly binds DNA and STING via its DEAD domain and triggers activation of signaling mediated by mitogen-activated protein kinases TBK1 and transcription factor IRF3, resulting IFN production (Zhang et al., 2011). DDX41 can also detect bacterial secondary messengers like cyclic di-GMP (c-di-GMP) and cyclic di-AMP (c-di-AMP), leading to formation of a complex with STING. This complex transmits the signal of bacterial intrusion to TBK1-IRF3 and activates the interferon response (Parvatiyar et al., 2012). Phosphorylation of Tyr414 of DDX41 is a pre-requisite for foreign dsDNA recognition and recruitment of STING. Besides, BTK’s kinase domain can bind the DEAD domain of DDX41 (Lee et al., 2015). After immune response, DDX41 will be ubiquitinated by TRIM21 through K48-mediated linkage for degradation. The ubiquitination sites are Lys9 and Lys115 (Zhang et al., 2013). Somatic DDX41 mutations have been reported in the study of sporadic acute myeloid leukemia (AML) syndrome (Ding et al., 2012). A familial MDS/ AML syndrome characterized by long latency and germline mutations in DDX41 gene is also identified (Polprasert et al., 2015). DDX41 can associate with spliceosomal proteins, and its defects lead to loss of tumor suppressor function due to altered pre-mRNA splicing and RNA processing (Polprasert et al., 2015). Although DDX41 plays important roles in innate immunity and diseases, the precise mechanism as well as the extent of involvement the protein in these processes is poorly understood. Here, we report the crystal structure of human DDX41 (hDDX41) DEAD domain complexed with an SO4 2− and an Mg to 2.26 Å resolution. There are strong interactions between different motifs to stabilize the whole structure. The P-loop presents in a half-open conformation. The DEAD domain protein can bind ADP and AMP but not ATP in vitro because of the steric hindrance. Most mutated amino acids related with familial MDS/AML are conserved. In addition, the N-terminal disordered region (amino acid 1–152) is shown targeting hDDX41 protein to the nucleus. Our study provides basic structural information for further researches on hDDX41 biological function and valuable insights for the treatment of DDX41-related diseases in the future. The crystal structure of hDDX41 DEAD domain was solved by molecular replacement using the structure of DDX5 domain I (PDB code: 3FE2) as the search model and refined to 2.26 Å resolution with an R factor of 0.19 (Rfree = 0.23). Details of data collection and refinement statistics are listed in Table S1. The crystal used for data collection belonged to space group P21. One asymmetric unit consists of two molecules of the protein based on the calculated solvent content of 44.15%. The hDDX41 DEAD domain consists of an α/β fold, which is similar to those observed for other members of the DEAD-box proteins for which structures are available. The overall structure consists of ten α-helices (α1–α10) and a β-sheet formed by eight β-

Structural and functional analyses of human DDX41 DEAD domain