Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme uptake

Paweł P Knejski, Satchal K Erramilli, Anthony A Kossiakoff

bioRxiv. 2023 Aug 1:2023.08.01.551527.

PMID: 37577460 PMCID: PMC10418163 DOI: 10.1101/2023.08.01.551527

Pathogenic bacteria, such as Pseudomonas aeruginosa , depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR’s heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.