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.