Structural basis for selective thymidine binding by the Borrelia burgdorferi substrate-binding protein BmpA

Area of Science:

• Structural biology
• Microbiology
• Biochemistry

Background:

• Borrelia burgdorferi, the Lyme disease agent, has limited metabolic capabilities and relies on salvage pathways for nucleotide synthesis.
• Understanding nutrient acquisition is vital for this pathogen and other organisms with reduced genomes.

Purpose of the Study:

• To determine the crystal structure of BmpA, a putative substrate-binding protein from Borrelia burgdorferi.
• To investigate the nucleoside-binding specificity and mechanism of BmpA.
• To identify evolutionarily conserved protein scaffolds involved in nucleoside uptake.

Main Methods:

• X-ray crystallography to determine BmpA structure at 2.6 Å resolution.
• Microscale thermophoresis to assess BmpA binding affinity for various nucleosides.
• Structure-guided mutagenesis to identify key residues for ligand recognition.
• Foldseek for structural homology searches across bacterial and archaeal species.

Main Results:

• The crystal structure revealed a conserved substrate-binding protein fold with a buried nucleoside-binding pocket.
• BmpA exhibits high-affinity binding for thymidine, followed by cytidine and adenosine; no binding was detected for ribose, guanosine, inosine, or uridine.
• Mutagenesis identified two conserved aromatic residues (Phe27 and Phe176) critical for thymidine recognition.
• Structural homology searches identified related proteins across diverse species, indicating an evolutionarily conserved scaffold for nucleoside binding.

Conclusions:

• Conserved binding protein architectures facilitate selective nucleoside acquisition in bacteria.
• BmpA's structure and binding properties provide insights into nutrient uptake strategies in organisms with reduced genomes.
• This research lays the groundwork for understanding pathogen metabolism and developing targeted interventions.