Abstract Dysreguated translation of tumor suppressor gene and/or oncogene mRNAs into proteins plays a crucial role in tumorigenesis. Translation initiation in mammalian cells is the rate-limiting step of mRNA translation/protein synthesis and primarily exerted through the eukaryotic translation initiation factor 4 F (eIF4F) complex consists of three subunits: eIF4E, which binds the cap of mRNA; eIF4A, an RNA helicase implicated in unwinding mRNA structure; and a scaffold protein eIF4G.The helicase activity of free eIF4A is low but it is dramatically stimulated by forming a complex with eIF4G although the molecular mechanisms are not completely understood. The crystal structure of the yeast eIF4A-eIF4G complex and the mapped interaction sites of eIF4AI-eIF4GII complex from nuclear magnetic resonance (NMR) studies provide insights into structural basis for eIF4G-enhanced eIF4A helicase activity. However, the atomic structure of human eIF4A-eIF4G complex and the biological function of protein-protein interaction for enhancing eIF4A helicase activity in tumor development have not been elucidated. Utilizing X-ray crystallography, we reported here the structure of the complex formed by human eIF4GI (residues 722-999) in a complex with human eIF4A1 (residues 20-406) at 2.8 Å resolution. eIF4A1 protein crystallized in an open, dumbbell-shape conformation with the linker between the two RecA-like domains. eIF4G1 contains 10 α-helices that form a right-handed solenoid and its convex surface interacts with both N- and C-terminal domains of eIF4A (eIF4A1-NTD and eIF4A1-CTD). A total buried surface area from both the proteins at the two interfaces measures ~2700 Å2, with an extensive contact area between the eIF4A1-CTD and the N-terminal portion of eIF4G1 and a secondary, much smaller interface involves eIF4A1-NTD and C-terminal portion of eIF4G1. Utilizing site-directed mutagenesis coupled with biochemical analyses, we identified R324 as a previously uncharacterized residue of eIF4A1 which when mutated completely abolished the interaction with eIF4G1. Biological studies in cancer cells showed that exogenous expression of mutant eIF4A1R324 in human prostate cancer cells significantly inhibited cell proliferation in vitro and attenuated tumor growth of xenografted human prostate cancer cells in immune deficient mice. Given that eIF4A1 helicase activity plays an important role in selectively controlling oncogenes mRNA translation in human cancer cells, our findings from structural and biological studies will lay the groundwork for the development of novel anti-cancer therapeutics targeting the eIF4A1-eIF4G1 interaction to block eIF4A1 helicase activity-driven oncogene protein synthesis and ultimately to inhibit tumor development. Citation Format: Puja Singh, Hanyong Chen, Young-In Chi, Junxuan Lu, Zigang Dong, Yibin Deng. Structural basis of human translation initiation factor eIF4G-enhanced helicase activity of eIF4A in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4447.