Biochemical insights into Energy Coupling Factor (ECF) transporters and exploration of the inhibitory potential of novel binders

ABC transporters, ubiquitous across life, utilize ATP hydrolysis to translocate diverse substrates across membranes via conserved cytoplasmic nucleotide-binding domains. Their Type III subclass (energy coupling factor transporters, ECF transporters), exclusive to prokaryotes, employs distinct micronutrient uptake mechanisms. This thesis investigates ECF transporters' structure, mechanism, and potential as novel antimicrobial targets.<br/>PdxU2, the substrate binding domain for microbial vitamin B6 uptake in ECF transporters, was cloned from a deep sea hyperthermophilic archaeon Pyrococcus abyssi and purified using detergent to main stability. A low-resolution structure (4.6 Å) was obtained, but ligand binding remained unresolved.<br/>Mechanistic studies performed with ECF-PanT and ECF-FolT2 from Lactobacillus delbrueckii which transport vitamin B5 and B9 and reconstituted in proteoliposomes, showed that the transporters require ATP hydrolysis, and are not very sensitive to pH but have significant thermal sensitivity, indicating substantial conformational rearrangements during catalysis.<br/>ECF transporters are exclusive to prokaryotes, including pathogens, and are a hitherto underexplored drug target. A systematic structure-activity relationship (SAR) study of ECF inhibitors identified key inhibition determinants. We confirmed our inhibitors' antimicrobial activity against the human pathogen S. pneumoniae and demonstrated effectiveness correlates with ECF transporter expression, establishing them as novel druggable targets against antimicrobial resistance.<br/>Nanobodies (Nbs), featuring monomeric architecture and high-affinity specificity, are powerful structural tools. We generated nanobodies against ECF-PanT. Biochemical validation confirmed binding, and proteoliposome uptake assays demonstrated Nb inhibition of ATP-dependent transport. Two Nbs exhibited nanomolar affinity for ECF-FolT2. Cryo-EM structures of Nb-ECF-FolT2 in detergent micelles revealed Nb binding sites on nucleotide binding domain (EcfA), and the complexes were found in the apo-conformation.