FeoC from Klebsiella pneumoniae uses its iron sulfur cluster to regulate the GTPase activity of the ferrous iron channel

Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe²⁺). The Feo system is crucial for colonization and virulence of pathogens. In γ-proteobacteria, the system consists of FeoA, FeoB, and FeoC. The function of FeoA remains unknown. FeoB likely forms the channel, whose regulation has been suggested to involve its GTPase domain (part of its NFeoB domain). FeoC from Klebsiella pneumonia was found to contain a [4Fe4S] cofactor, whose presence was speculated to enhance the GTPase activity of FeoB (Hsueh, K.-L., et al., J. Bacteriol. 2013 195(20): 4726–34). We present results here that support and extend that hypothesis. We monitored the GTPase activity of FeoB by NMR spectroscopy and found that the presence of 7% FeoC-[4Fe-4S]³⁺ (the highest level of cofactor achieved in vitro) increased the GTPase rate of NFeoB by 3.6-fold over NFeoB. The effect depends on the oxidation state of the cluster; with reduction of the cluster to [4Fe-4S]²⁺ the GTPase greatly decreased the GTPase rate. From the effects of point mutations in FeoC on GTPase rates, we conclude that Lys62 and Lys68 on FeoC each contribute to increased GTPase activity on NFeoB. Mutation of Thr37 of NFeoB to Ser nearly abolished the GTPase activity. The GTPase activity of the isolated K. pneumoniae NFeoB-FeoC complex (NFeoBC) was found to be higher in KCl than in NaCl solution. We solved the X-ray structure of the NFeoBC crystallized from KCl and compared it with a prior X-ray structure crystalized from NaCl. We propose a hypothesis, consistent with these results, to explain the factors that influence the GTPase activity. Bacteria may use the oxygen-sensitive cluster as a sensor to up-regulate the gate closing speed.     

Structure of the GTPase and GDI domains of FeoB, the ferrous iron transporter of Legionella pneumophila

Prokaryotic pathogens have developed specialized mechanisms for efficient uptake of ferrous iron (Fe²⁺) from the host. In Legionella pneumophila, the causative agent of Legionnaires’ disease, the transmembrane GTPase FeoB plays a key role in Fe²⁺ acquisition and virulence. FeoB consists of a membrane-embedded core and an N-terminal, cytosolic region (NFeoB). Here, we report the crystal structure of NFeoB from L. pneumophila, revealing a monomeric protein comprising two separate domains with GTPase and guanine-nucleotide dissociation inhibitor (GDI) functions. The GDI domain displays a novel fold, whereas the overall structure of the GTPase domain resembles that of known G domains but is in the rarely observed nucleotide-free state.     

A general protocol for the expression and purification of the intact transmembrane transporter FeoB

Ferrous iron (Fe²⁺) transport is an essential process that supports the growth, intracellular survival, and virulence of several drug-resistant pathogens, and the ferrous iron transport (Feo) system is the most important and widespread protein complex that mediates Fe²⁺ transport in these organisms. The Feo system canonically comprises three proteins (FeoA/B/C). FeoA and FeoC are both small, accessory proteins localized to the cytoplasm, and their roles in the Fe²⁺ transport process have been of great debate. FeoB is the only wholly-conserved component of the Feo system and serves as the inner membrane-embedded Fe²⁺ transporter with a soluble G-protein-like N-terminal domain. In vivo studies have underscored the importance of Feo during infection, emphasizing the need to better understand Feo-mediated Fe²⁺ uptake, although a paucity of research exists on intact FeoB. To surmount this problem, we designed an overproduction and purification system that can be applied generally to a suite of intact FeoBs from several organisms. Importantly, we noted that FeoB is extremely sensitive to excess salt while in the membrane of a recombinant host, and we designed a workflow to circumvent this issue. We also demonstrated effective protein extraction from the lipid bilayer through small-scale solubilization studies. We then applied this approach to the large-scale purifications of Escherichia coli and Pseudomonas aeruginosa FeoBs to high purity and homogeneity. Lastly, we show that our protocol can be generally applied to various FeoB proteins. Thus, this workflow allows for isolation of suitable quantities of FeoB for future biochemical and biophysical characterization.     

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.     

The FeoA protein is necessary for the FeoB transporter to import ferrous iron

In many bacterial feo loci, the feoA gene is associated with the feoB gene. While the feoB-encoded FeoB protein has been demonstrated as a ferrous iron [Fe(II)] transporter, the function of the feoA gene product, FeoA, is unknown. In the present study, we report that the FeoA protein interacts with the FeoB Fe(II) transporter, which is required for FeoB-mediated Fe(II) uptake in Salmonella enterica. Iron uptake assay revealed that in the absence of FeoA, FeoB import of Fe(II) is impaired. Bacterial two-hybrid assay determined that the FeoA protein directly and specifically binds to the FeoB transporter in vivo. This FeoA-FeoB interaction appeared necessary for FeoB-mediated Fe(II) uptake because Salmonella expressing the mutant FeoA that cannot interact with FeoB failed to uptake Fe(II) via the FeoB transporter. Finally, we showed that the FeoA protein does not affect expression of the FeoB transporter per se.     

Structure and Function of the FeoB G-Domain from Methanococcus jannaschii

FeoB in bacteria and archaea is involved in the uptake of ferrous iron (Fe²⁺), an important cofactor in biological electron transfer and catalysis. Unlike any other known prokaryotic membrane protein, FeoB contains a GTP-binding domain at its N-terminus. We determined high-resolution X-ray structures of the FeoB G-domain from Methanococcus jannaschii with and without bound GDP or Mg²⁺-GppNHp. The G-domain forms the same dimer in all three structures, with the nucleotide-binding pockets at the dimer interface, as in the ATP-binding domain of ABC transporters. The G-domain follows the typical fold of nucleotide-binding proteins, with a β-strand inserted in switch I that becomes partially disordered upon GTP binding. Switch II does not contact the nucleotide directly and does not change its conformation in response to the bound nucleotide. Release of the nucleotide causes a rearrangement of loop L6, which we identified as the G5 region of FeoB. Together with the C-terminal helix, this loop may transmit the information about the nucleotide-bound state from the G-domain to the transmembrane region of FeoB.     

A novel strategy for the identification of protein–DNA contacts by photocrosslinking and mass spectrometry

Photochemical crosslinking is a method for studying the molecular details of protein–nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe³⁺-IMAC (immobilized metal affinity chromatography) purification of peptide–oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein–DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide–oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe³⁺-IMAC. A combination of MS analysis of the peptide–nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys²⁰⁹ of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys²⁰⁹ is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.     

Lon-Mediated Proteolysis of the FeoC Protein Prevents Salmonella enterica from Accumulating the Fe(II) Transporter FeoB under High-Oxygen Conditions

The Salmonella Feo system consists of the FeoA, FeoB, and FeoC proteins and mediates ferrous iron [Fe(II)] import. FeoB is an inner membrane protein that, along with contributions from two small hydrophilic proteins, FeoA and FeoC, transports Fe(II). We previously reported that FeoC binds to and protects the FeoB transporter from FtsH-mediated proteolysis. In the present study, we report proteolytic regulation of FeoC that occurs in an oxygen-dependent fashion. While relatively stable under low-oxygen conditions, FeoC was rapidly degraded by the Lon protease under high-oxygen conditions. The putative Fe-S cluster of FeoC seemed to function as an oxygen sensor to control FeoC stability, as evidenced by the finding that mutation of the putative Fe-S cluster-binding site greatly increased FeoC stability under high-oxygen conditions. Salmonella ectopically expressing the feoB and feoC genes was able to accumulate FeoB and FeoC only under low-oxygen conditions, suggesting that FeoC proteolysis prevents Salmonella from accumulating the FeoB transporter under high-oxygen conditions. Finally, we propose that Lon-mediated FeoC proteolysis followed by FtsH-mediated FeoB proteolysis helps Salmonella to avoid uncontrolled Fe(II) uptake during the radical environmental changes encountered when shifting from low-iron anaerobic conditions to high-iron aerobic conditions.     

The FeoC Protein Leads to High Cellular Levels of the Fe(II) Transporter FeoB by Preventing FtsH Protease Regulation of FeoB in Salmonella enterica

In the gammaproteobacteria, the FeoA, FeoB, and FeoC proteins constitute the Feo system, which mediates ferrous iron [Fe(II)] import. Of these Feo proteins, FeoB is an inner membrane Fe(II) transporter that is aided by the small protein FeoA. However, the role of another small protein, FeoC, has remained unknown. Here we report that the FeoC protein is necessary for FeoB protein-mediated Fe(II) uptake in Salmonella experiencing low levels of oxygen and iron. The FeoC protein was found to directly bind to the FeoB transporter, leading to high cellular levels of FeoB. Depletion of the FtsH protease enabled high levels of FeoB in the absence of FeoC, suggesting that the FeoC protein protects the FeoB transporter from FtsH-mediated proteolysis. Our present study provides a singular example of bacteria that can control expression of iron uptake systems posttranslationally by employing a small iron transporter-binding protein.     

Modulation of receptor dynamics by the regulator of G protein signaling Sst2

G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.     

The initiation of GTP hydrolysis by the G-domain of FeoB: insights from a transition-state complex structure

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoB^St) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF₄ ⁻, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoB^St, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoB^St. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.     

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

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe²⁺) 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.     

Purification of MAP–kinase protein complexes and identification of candidate components by XL–TAP–MS

The purification of low-abundance protein complexes and detection of in vivo protein–protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL–TAP–MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL–TAP–MS to study the MKK2–Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde–crosslinked protein complexes under fully denaturing conditions. Combined with ¹⁵N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2–MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein–protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL–TAP–MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein–protein interactions.

XL–TAP–MS: a novel technique that allows purification of crosslinked, low abundant protein complexes from plant tissues under denatured conditions and detection of in vivo protein–protein interactions.     

Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: three questions

Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson’s disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein.     

Regulation of contractile vacuole formation and activity in Dictyostelium

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV–plasma membrane fusion and discharging. disgorgin⁻ cells produce large CVs due to impaired CV–plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin⁻ large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.     

Three‐dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6‐bladed β‐propeller hydrolase family

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe²⁺. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe²⁺. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca²⁺, Mg²⁺, or Zn²⁺ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe²⁺ as a catalytic metal. This could further expand our understanding of utilization of Fe²⁺ not only in oxidative enzymes but also in hydrolases.