ABSTRACT
Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn.
Subject(s)
Cation Transport Proteins/isolation & purification , Animals , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Hepcidins/metabolism , Hot Temperature/adverse effects , Mice , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Ferroportin (Fpn)-the only known cellular iron exporter-transports dietary and recycled iron into the blood plasma, and transfers iron across the placenta. Despite its central role in iron metabolism, our molecular understanding of Fpn-mediated iron efflux remains incomplete. Here, we report that Ca2+ is required for human Fpn transport activity. Whereas iron efflux is stimulated by extracellular Ca2+ in the physiological range, Ca2+ is not transported. We determine the crystal structure of a Ca2+-bound BbFpn, a prokaryotic orthologue, and find that Ca2+ is a cofactor that facilitates a conformational change critical to the transport cycle. We also identify a substrate pocket accommodating a divalent transition metal complexed with a chelator. These findings support a model of iron export by Fpn and suggest a link between plasma calcium and iron homeostasis.
Subject(s)
Calcium/chemistry , Cation Transport Proteins/chemistry , Animals , Binding Sites , Chelating Agents/chemistry , Crystallography, X-Ray , HEK293 Cells , Homeostasis , Humans , Iron/chemistry , Metals/chemistry , Mutagenesis , Oocytes/metabolism , Protein Conformation , XenopusABSTRACT
Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.
Subject(s)
Membrane Proteins/metabolism , Animals , Binding Sites , Blotting, Western , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Line , Ceruloplasmin/metabolism , Humans , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Protein Structure, SecondaryABSTRACT
In vertebrates, the iron exporter ferroportin releases Fe(2+) from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anaemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homologue of ferroportin, BbFPN, in both the outward- and inward-facing states. Despite undetectable sequence similarity, BbFPN adopts the major facilitator superfamily fold. A comparison of the two structures reveals that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identify a substrate metal-binding site, based on structural and mutational analyses. Furthermore, the BbFPN structures suggest that a predicted hepcidin-binding site of ferroportin is located within its central cavity. Thus, BbFPN may be a valuable structural model for iron homeostasis regulation by ferroportin.
Subject(s)
Bdellovibrio/metabolism , Cation Transport Proteins/metabolism , Binding Sites , Cation Transport Proteins/chemistry , Humans , Iron/metabolism , Protein Conformation , Structural Homology, ProteinABSTRACT
The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Guanosine Diphosphate/metabolism , Amino Acid Sequence , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine Diphosphate/chemistry , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.
Subject(s)
Cation Transport Proteins/metabolism , Escherichia coli Proteins/metabolism , Guanosine Diphosphate/metabolism , Nucleotides/metabolism , Amino Acid Sequence , Binding Sites/genetics , Calorimetry/methods , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Nucleotides/chemistry , Nucleotides/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , ThermodynamicsABSTRACT
GTPases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric Gαßγ proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the Gα subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a Gαs G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G protein-coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus, which includes an N-terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP-bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTPase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base.
Subject(s)
Bacterial Proteins/metabolism , GTP-Binding Proteins/metabolism , Guanosine Diphosphate/metabolism , Alanine/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalysis , Fluorescence , Humans , Hydrolysis , Molecular Sequence Data , Sequence Homology, Amino Acid , Serine/genetics , Streptococcus thermophilus/metabolismABSTRACT
FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe(2+) transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer.
Subject(s)
GTP Phosphohydrolases/chemistry , Gallionellaceae/enzymology , Membrane Proteins/chemistry , Protein Multimerization , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, ProteinABSTRACT
Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+ß and α/ß fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genes, Bacterial/genetics , Integrons/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Gene Transfer, Horizontal/genetics , Metagenome/genetics , Models, Molecular , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Protein Binding , Protein Structure, Secondary , Vibrio cholerae/genetics , Vibrio cholerae/metabolismABSTRACT
BACKGROUND: The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. METHODOLOGY/PRINCIPAL FINDINGS: We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. CONCLUSIONS/SIGNIFICANCE: Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.