Structural Characterization of Trypanosoma cruzi P5CS Filaments by Cryo-Electron Microscopy: Insights Into Proline Metabolism and Drug Discovery

• Grant number: 25/11958-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: October 13, 2025
• End date: July 30, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Otavio Henrique Thiemann
• Grantee: Maria Eduarda Souza Dias Lino
• Supervisor: Leandro Farias Estrozi
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Institution abroad: Institut de Biologie Structurale (IBS), France
• Associated to the scholarship: 23/15602-8 - Structural study of the enzymes P5C, P5CS, P5CR, P5CDH and PRODH of Proline metabolism in Trypanosoma cruzi as targets for the discovery of drugs against Chagas Disease, BP.DR

Abstract

Proline metabolism is essential for trypanosomatids to adapt to diverse environments duringtheir life cycle. In Trypanosoma cruzi, the enzyme ¿¹-pyrroline-5-carboxylate synthase(TcP5CS) is crucial for proline biosynthesis and comprises two functional domains:glutamate kinase (TcGK) and glutamyl phosphate reductase (TcGPR). TcP5CS homologueshave been shown to form filamentous assemblies, potentially linked to enzymatic regulationand function. Recent findings from our laboratory revealed that TcP5CS also formsfilaments, displaying a structural organization distinct from its homologues. This project aimsto elucidate the three-dimensional structure of TcP5CS filaments and its domains usingsingle-particle cryo-electron microscopy (cryo-EM). Cryo-EM grids will be analyzed underdifferent biochemical conditions. The TcGK and TcGPR domains are also being crystallizedfor structural determination by X-ray crystallography, providing complementary atomic-resolution data. Biophysical techniques such as circular dichroism (CD), multi-angle lightscattering (SEC-MALS), and small-angle X-ray scattering (SAXS) are being applied to betterunderstand filament formation and enzyme architecture. The internship will take place in thelaboratory of Dr. Leandro Farias Estrozi, CNRS - France, from October to July 2026. Theexpertise acquired in cryo-EM and crystallography will be transferred to Brazilian researchgroups, contributing to the structural and functional characterization of TcP5CS andsupporting future drug discovery strategies targeting proline metabolism in T. cruzi, a keypathway for parasite survival. (AU)