Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.
| Autor: | Knejski PP; Deparment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.; Laboratory of Medical Biology, Faculty of Biotechnology, University of Wrocław, Wrocław 50-383, Poland.; Present address: Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich 8093, Switzerland., Erramilli SK; Deparment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.; Present address: Meso Scale Diagnostics, LLC, Rockville, Maryland 20850, USA., Kossiakoff AA; Deparment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.; Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA. |
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| Jazyk: | angličtina |
| Zdroj: | BioRxiv : the preprint server for biology [bioRxiv] 2024 Aug 05. Date of Electronic Publication: 2024 Aug 05. |
| DOI: | 10.1101/2023.08.01.551527 |
| Abstrakt: | 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 Interests: DECLARATION OF INTEREST The authors declare no competing interests. |
| Databáze: | MEDLINE |
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