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TitleChaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.
Journal, issue, pagesStructure, Vol. 32, Issue 4, Page 411-423.e6, Year 2024
Publish dateApr 4, 2024
AuthorsPaweł P Knejski / Satchal K Erramilli / Anthony A Kossiakoff /
PubMed AbstractPathogenic 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 ...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.
External linksStructure / PubMed:38325368 / PubMed Central
MethodsEM (single particle)
Resolution2.5 Å
Structure data

41255

8the

EMDB-41255, PDB-8the:
Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transporter PhuR in complex with synthetic antibody and heme
Method: EM (single particle) / Resolution: 2.5 Å

Chemicals

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

ChemComp-CTQ:
(2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid

ChemComp-HOH:
WATER / Water

Source
  • pseudomonas aeruginosa (bacteria)
  • homo sapiens (human)
KeywordsMEMBRANE PROTEIN / beta barrel / outer membrane protein / heme binding protein / heme transport

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