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Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)
Published on: November 29, 2014
The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with
Damon S Anderson1, Pratima Adhikari, Andrew J Nowalk
1Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Lothrop St., Pittsburgh, PA 15261, USA.
Abstract:
Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.
Insights
This study reveals how the Haemophilus influenzae FbpABC iron transporter functions in Escherichia coli, showing its specificity for ferric iron (Fe3+) and dependence on the FbpA binding protein for transport.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Pathogenic bacteria like Haemophilus influenzae use FbpABC iron transporters to move iron from the periplasm to the cytosol.
- Understanding these transporters is crucial for developing new antimicrobial strategies.
Purpose of the Study:
- To biochemically characterize the Haemophilus influenzae FbpABC iron transporter in a siderophore-deficient Escherichia coli model.
- To elucidate the transport mechanism, substrate specificity, and the role of the FbpA binding protein.
Main Methods:
- Radiolabeled Fe3+ transport assays to determine kinetic parameters (Km, Vmax).
- ATPase and protonmotive force inhibitor studies to assess energy dependence.
- Metal competition and sensitivity experiments to evaluate substrate specificity and toxicity.
- Site-directed mutagenesis of the FbpA binding protein to assess iron binding affinity and transport activity.
Main Results:
- FbpABC-mediated transport exhibited an apparent Km of 0.9 microM and Vmax of 1.8 pmol/10(7)cells/min.
- Transport was dependent on the FbpA binding protein and inhibited by ATPase inhibitors, but not protonmotive force inhibitors.
- The transporter showed high specificity for Fe3+ and selectivity for trivalent metals (Ga3+, Al3+) over divalent metals.
- A mutation in FbpA (Y196I) significantly reduced iron binding affinity but the mutant transporter retained substantial activity (35% of wild-type).
- Gallium-induced toxicity was observed specifically in E. coli expressing FbpABC.
Conclusions:
- The FbpABC transporter complex shares characteristics with bacterial binding protein-dependent ABC transporters.
- High specificity and affinity for ferric iron suggest a specialized role in iron acquisition.
- The FbpABC transporter is essential for satisfying the strict chemical requirements of Fe3+ binding and transport in bacteria.
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