High-Confidence Structural Model of the Trypanosoma cruzi GP63-like Metalloprotease Generated by AlphaFold and Validated through Molecular Dynamics Simulations

Abstract

The 63 kDa glycoprotein (TcGP63) is the major surface metalloprotease of Trypanosoma cruzi and plays a central role in cell invasion and immune evasion processes. Despite its biological relevance, the three-dimensional structure of this enzyme has not yet been experimentally determined. In this study, TcGP63 was modeled using AlphaFold, and the obtained model was evaluated for stereochemical, energetic, and conformational quality. The predicted structure presented high confidence values (pTM = 0.93; pLDDT > 90), indicating the robustness and accuracy of the model. The conserved catalytic motif HE XX H and the coordinated Zn²⁺ ion were clearly identified, with average coordination distances compatible with other metalloproteases of the metzincin family. Dynamic validation was conducted through 25 ns simulations using the GROMOS54a7 force field. Analyses of RMSD, radius of gyration (Rg), and RMSF demonstrated conformational stability and structural compactness throughout the simulation, with fluctuations localized only at the C-terminal extremity. Collectively, the results confirm the structural integrity of TcGP63 and provide a solid basis for molecular docking studies and the rational design of inhibitors aimed at controlling Chagas Disease.