RT Journal Article
SR Electronic
T1 Chaperone-assisted cryo-EM structure of <em>P. aeruginosa</em> PhuR reveals molecular basis for heme binding
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.08.01.551527
DO 10.1101/2023.08.01.551527
A1 Knejski, Paweł P.
A1 Erramilli, Satchal K.
A1 Kossiakoff, Anthony A.
YR 2024
UL http://biorxiv.org/content/early/2024/08/05/2023.08.01.551527.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.