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Sci Rep. 2019 Apr 30;9(1):6705. doi: 10.1038/s41598-019-42857-9.

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.

Author information

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.
3
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. dufaum@mail.nih.gov.

Abstract

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 findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified 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 effects 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 affect PKA catalytic efficiency. Inhibition of phosphorylation may be achieved by small, drug-like molecules that bind to GRTH and reconfigure the GRTH/PKA interface.

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