Biochemical and structural studies of the human DDX3X protein

• Grant number: 25/04023-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2025
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Ivan Rosa e Silva
• Grantee: Eric Leandro Lima Mendonça
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

The enzyme DDX3X is an ATP-dependent RNA helicase from the DEAD-box family that plays a crucial role in RNA metabolism, particularly in neurodevelopmental processes. This protein is composed of two RecA-like domains that form the ATP and RNA binding regions, flanked by flexible N- and C-terminal ends. The hypothesis of this work is that these terminal regions may regulate the enzymatic functions of DDX3X through their interaction with the structured domain. Therefore, the overall objective of this work is to study the structure-function relationship of the protein truncated at the N-terminal region using protein crystallography and enzymatic assays. The research group has already established protocols for the expression, purification, and crystallization of truncated constructs of the DDX3X protein and will provide samples of purified recombinant protein for the execution of this project. The enzymatic activity of the protein will be studied using UV absorption spectroscopy of the product of the ATPase enzymatic reaction in a coupled assay. We will optimize initial crystallization conditions, and the crystals will be subjected to X-ray diffraction at the Manacá-Sirius beamline (LNLS-CNPEM). The structure of the truncated protein will be determined by molecular replacement and refined. The results of this project will enable the elucidation of the molecular mechanisms regulating the function of the DDX3X protein. (AU)