Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu
We studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt)...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1355253/full |
| _version_ | 1797239011050258432 |
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| author | Taihao Yang Ye Zou Ho Leung Ng Ashish Kumar Salete M. Newton Phillip E. Klebba |
| author_facet | Taihao Yang Ye Zou Ho Leung Ng Ashish Kumar Salete M. Newton Phillip E. Klebba |
| author_sort | Taihao Yang |
| collection | DOAJ |
| description | We studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt) and glucosylated enterobactin (FeGEnt), their dicatecholate degradation product Fe(DHBS)2 (FeEnt*), the monocatecholates dihydroxybenzoic acid (FeDHBA) and dihydroxybenzoyl serine (FeDHBS), and the siderophore antibiotics cefiderocol (FDC) and MB-1. Unlike high-affinity ligand-gated porins (LGPs), Fiu-FM had only micromolar affinity for iron complexes. Its apparent KD values for FeDHBS, FeDHBA, FeEnt*, FeEnt, FeGEnt, FeFDC, and FeMB-1 were 0.1, 0.7, 0.7, 1.0, 0.3, 0.4, and 4 μM, respectively. Despite its broad binding abilities, the transport repertoires of E. coli Fiu, as well as those of Cir and FepA, were less broad. Fiu only transported FeEnt*. Cir transported FeEnt* and FeDHBS (weakly); FepA transported FeEnt, FeEnt*, and FeDHBA. Both Cir and FepA bound FeGEnt, albeit with lower affinity. Related transporters of Acinetobacter baumannii (PiuA, PirA, BauA) had similarly moderate affinity and broad specificity for di- or monomeric ferric catecholates. Both microbiological and radioisotopic experiments showed Fiu’s exclusive transport of FeEnt*, rather than ferric monocatecholate compounds. Molecular docking and molecular dynamics simulations predicted three binding sites for FeEnt*in the external vestibule of Fiu, and a fourth site deeper in its interior. Alanine scanning mutagenesis in the outermost sites (1a, 1b, and 2) decreased FeEnt* binding affinity as much as 20-fold and reduced or eliminated FeEnt* uptake. Finally, the molecular dynamics simulations suggested a pathway of FeEnt* movement through Fiu that may generally describe the process of metal transport by TonB-dependent receptors. |
| first_indexed | 2024-04-24T17:44:45Z |
| format | Article |
| id | doaj.art-251b280807874e81b191f2004fc73118 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-24T17:44:45Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-251b280807874e81b191f2004fc731182024-03-27T14:15:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-03-011510.3389/fmicb.2024.13552531355253Specificity and mechanism of TonB-dependent ferric catecholate uptake by FiuTaihao YangYe ZouHo Leung NgAshish KumarSalete M. NewtonPhillip E. KlebbaWe studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt) and glucosylated enterobactin (FeGEnt), their dicatecholate degradation product Fe(DHBS)2 (FeEnt*), the monocatecholates dihydroxybenzoic acid (FeDHBA) and dihydroxybenzoyl serine (FeDHBS), and the siderophore antibiotics cefiderocol (FDC) and MB-1. Unlike high-affinity ligand-gated porins (LGPs), Fiu-FM had only micromolar affinity for iron complexes. Its apparent KD values for FeDHBS, FeDHBA, FeEnt*, FeEnt, FeGEnt, FeFDC, and FeMB-1 were 0.1, 0.7, 0.7, 1.0, 0.3, 0.4, and 4 μM, respectively. Despite its broad binding abilities, the transport repertoires of E. coli Fiu, as well as those of Cir and FepA, were less broad. Fiu only transported FeEnt*. Cir transported FeEnt* and FeDHBS (weakly); FepA transported FeEnt, FeEnt*, and FeDHBA. Both Cir and FepA bound FeGEnt, albeit with lower affinity. Related transporters of Acinetobacter baumannii (PiuA, PirA, BauA) had similarly moderate affinity and broad specificity for di- or monomeric ferric catecholates. Both microbiological and radioisotopic experiments showed Fiu’s exclusive transport of FeEnt*, rather than ferric monocatecholate compounds. Molecular docking and molecular dynamics simulations predicted three binding sites for FeEnt*in the external vestibule of Fiu, and a fourth site deeper in its interior. Alanine scanning mutagenesis in the outermost sites (1a, 1b, and 2) decreased FeEnt* binding affinity as much as 20-fold and reduced or eliminated FeEnt* uptake. Finally, the molecular dynamics simulations suggested a pathway of FeEnt* movement through Fiu that may generally describe the process of metal transport by TonB-dependent receptors.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1355253/fullsiderophorecatecholateiron transportTonBfluorescent sensorsite-directed mutagenesis |
| spellingShingle | Taihao Yang Ye Zou Ho Leung Ng Ashish Kumar Salete M. Newton Phillip E. Klebba Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu Frontiers in Microbiology siderophore catecholate iron transport TonB fluorescent sensor site-directed mutagenesis |
| title | Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu |
| title_full | Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu |
| title_fullStr | Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu |
| title_full_unstemmed | Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu |
| title_short | Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu |
| title_sort | specificity and mechanism of tonb dependent ferric catecholate uptake by fiu |
| topic | siderophore catecholate iron transport TonB fluorescent sensor site-directed mutagenesis |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1355253/full |
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