Structural and Functional Analyses of Trypanosoma brucei Nucleoside Diphosphate Kinase

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), relies exclusively on purine salvage for nucleotide biosynthesis, making its nucleotide-processing enzymes attractive drug targets. Here, we present a comprehensive structural and functional characterization of T. brucei's nucleoside diphosphate kinase B (TbNDPK), a key enzyme in nucleotide homeostasis. Circular dichroism and fluorescence spectroscopy revealed that TbNDPK is highly stable under thermal and chemical stress and undergoes nucleotide-induced conformational changes. This study also presents high-resolution crystal structures of the apo enzyme and complexes with UDP, CDP, and GDP, showing a conserved hexameric fold, with induced-fit binding via a flexible loop involving Phe59 and key active-site residues. Enzymatic assays revealed substrate preferences for UDP and GDP, while deoxyribonucleotide diphosphates were processed with significantly reduced efficiency. Molecular dynamics simulations revealed ligand-dependent flexibility and subunit-specific nucleotide dynamics, indicating potential asymmetry and cooperative communication within the hexamer. Collectively, these findings position TbNDPK as a thermostable, catalytically efficient, and structurally distinct enzyme optimized for ribonucleotide metabolism and support its potential as a selective target for future antitrypanosomal drug discovery.