Abstact

BmpA is a putative substrate-binding protein from Borrelia burgdorferi, the causative agent of Lyme disease, an organism with limited metabolic capacity that relies on salvage pathways rather than de novo nucleotide biosynthesis. Here, we determine the crystal structure of BmpA to a resolution of 2.6 A, revealing a conserved substrate-binding protein fold with a deeply buried nucleoside-binding pocket. Using microscale thermophoresis, we show that BmpA binds thymidine with high affinity followed by cytidine and adenosine, whereas binding to ribose, guanosine, inosine, and uridine was not detected. Structure-guided mutagenesis further demonstrates that two conserved aromatic residues (Phe27 and Phe176) are essential for thymidine recognition, as alanine substitution at either position abolishes detectable binding. Additionally, a Foldseek-based structural homology search identified related proteins across diverse bacterial and archaeal species that share a conserved overall fold and binding-site architecture despite low sequence similarity, consistent with an evolutionarily conserved scaffold that can accommodate distinct nucleoside ligands. Together, our work illustrates how conserved binding protein architectures enable selective nucleoside acquisition and provides a foundation for understanding nutrient uptake strategies in organisms with reduced genomes.