1 SCIENTIFIC REPORTS | (2019) 9:6705 | https://doi.org/10.1038/s41598-019-42857-9 www.nature.com/scientificreports Characterization of the Phosphorylation Site of GRTH/ DDX25 and Protein Kinase A Binding Interface Provides Structural Basis for the Design of a Non-Hormonal Male Contraceptive Murugananthkumar Raju 1 , Sergio A. Hassan 2 , Raghuveer Kavarthapu 1 , Rajakumar Anbazhagan 1 & Maria L. Dufau 1 Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8% of Japanese patient population with azoospermia. Transfection of the mutant GRTH construct in COS-1 cells leads to loss of the 61 kDa cytoplasmic phospho-species. Mice with knock-in of the human GRTH mutation are sterile and lack sperm with normal androgen and mating behavior. These fndings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specifc phospho-antibody, we have identifed T239, structurally adjacent to the patient’s mutant site as the GRTH phospho-site. Molecular modelling provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/ PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These efects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA interface, which leads to modest but consequential structural changes that can afect PKA catalytic efciency. Inhibition of phosphorylation may be achieved by small, drug-like molecules that bind to GRTH and reconfgure the GRTH/PKA interface. Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is a member of the DEAD-box family of RNA helicases identifed and isolated from rat, mouse, and human testes 1,2 . Tis testis-specifc protein expressed in Leydig and germ cells of the male gonad is transcriptionally upregulated by gonadotropin through direct and indirect actions of androgen via androgen receptor, respectively 3–5 . In Leydig cells, GRTH regulates cholesterol homeostasis via its action on StAR expression 6 . In germ cells, it is expressed predominantly in meiotic spermat- ocytes and round spermatids where is essential for spermatid development and completion of spermatogenesis. GRTH knockout mice lack sperm due to failure of round spermatids to elongate at step 8 of spermiogenesis 7 . Tis multifunctional protein participates in the export of relevant mRNAs from nucleus to cytoplasmic sites of germ cells for storage/degradation in chromatoid bodies of round spermatids and translation, as indicated through its association with actively translating polyribosomes in germ cells 8,9 . It was also found to be a regulator of miRNA biogenesis 10 and an inhibitor of germ cell apoptosis 11 . 1 Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, OIR/CIT, National Institutes of Health, Bethesda, MD, 20892-4510, USA. 2 Center for Molecular Modeling, OIR/CIT, National Institutes of Health, Bethesda, MD, 20892-4510, USA. Murugananthkumar Raju and Sergio A. Hassan contributed equally. Correspondence and requests for materials should be addressed to M.L.D. (email: dufaum@mail.nih.gov) Received: 6 August 2018 Accepted: 10 April 2019 Published: xx xx xxxx OPEN