Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

Hung, Kevin; Chang, Yi‐Wei; Eng, Edward T.; Chen, Jai Hui; Chen, Yi-Chung; Sun, Yuh‐Chang; Hsiao, Chwan‐Deng; Dong, Gang; Spasov, Krasimir A.; Unger, Vinzenz M.; Huang, Tai Huang

doi:10.1016/j.jsb.2010.01.017

Cited by 31 publications

(35 citation statements)

References 45 publications

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“…Prior to our study, several lines of evidence suggested that the Feo proteins may interact to form a functional Feo structure. Interactions among FeoB monomers have been suggested by crystallography studies (51)(52)(53)(54). An interaction between FeoC and FeoB was observed in a BACTH assay for both the V. cholerae (27) and S. enterica (41) proteins.…”

Section: Discussionmentioning

confidence: 94%

“…Further, significant limitations to studying Feo structure by crystallography have been observed, since a consensus on the oligomeric state of FeoB has never been reached. For example, FeoB N-terminal domains from several organisms have crystallized as monomers (35,53,(55)(56)(57) whereas the FeoB N-terminal domains from other organisms have crystallized as dimers or trimers (51)(52)(53)(54). Since those studies used a truncated FeoB protein, interactions involving the membrane-spanning regions or C terminus of FeoB were not detected.…”

Section: Discussionmentioning

confidence: 99%

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Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex

2016

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Feo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. The feo operon in Vibrio cholerae is made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linked in vivo in V. cholerae. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport. IMPORTANCEThe Feo system is the major route for ferrous iron transport in bacteria. In this work, the Vibrio cholerae Feo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complex in vivo. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly. Iron is an indispensable component of enzymes involved in a wide range of biological processes and, as a result, is essential for life in almost all organisms (1). Notwithstanding the fact that iron is abundant in nature, aerobic, neutral-pH environments favor the formation of Fe(OH) 3 , a highly insoluble ferric iron (Fe 3ϩ ) complex. In contrast, in anoxic environments, free ferrous iron (Fe 2ϩ ) is more readily available (2). In order to meet their iron requirement, bacteria have multiple iron transport systems that allow use of the various forms of iron present in their environment, and Vibrio cholerae is no exception (3).V. cholerae is a human pathogen that causes cholera, a severe diarrheal disease resulting from the ingestion of contaminated food or water (4). V. cholerae must acquire iron from different environments, such as the gut of its human host or the fresh, brackish, and ocean waters that make up its natural habitat. This need to acquire the various forms of iron is reflected in the observation that approximately 1% of its genome is devoted to the acquisition of iron (3). In V. cholerae, ferric iron is commo...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex

2016

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“…NFeoB consists of a small-GTPase domain (G domain) and a GDP dissociation inhibitor (GDI)-like helical domain. The crystal structures of several forms from E. coli and Klebsiella pneumoniae NFeoB assemble into a funnel-like trimer with a cytoplasmic pore that could facilitate gating and passage of unhydrated ferrous irons (17,18). The G domain exhibits guanosine nucleotide-dependent conformational changes in the Switch I, Switch II, and G5 motifs of the G domain and changes in the distance between G and GDI-like domains.…”

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confidence: 99%

“…For preparing the holo-FeoC, the protein was purified through the GST column and concentrated aerobically, whereas the follow-up enzyme digestion was performed anaerobically (less than 5 ppm oxygen) in an anaerobic chamber from COY Lab (Grass Lake, MI) and purified through a Superdex-75 10/300 column attached to an ÄKTA purifier (GE Healthcare) inside the anaerobic chamber. Samples were kept at low temperature in a labtop cooler from Nalgene/Sigma-Aldrich (St. Louis, MO 3 and 1% (vol/vol) BME vitamins, as described previously (18,19,21), but with modifications: an additional 50 mg/liter FeCl3 (wt/vol) and 1% (vol/vol) BME vitamins were added to the growth medium. For preparing [U- 13 C, 15 N]-KpFeoC protein, [U-…”

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confidence: 99%

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Hsueh

Chen

et al. 2013

J Bacteriol

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Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe 2؉ ) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen-or iron-sensitive coordination of the Fe-S cluster.

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“…Notably, the structures of NFeoB from several species have been solved (3,11,13,14,19,27). We previously determined the Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus NFeoB crystal structures with and without bound ligands (14).…”

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confidence: 99%