Bacterial Heme Transport Research Articles

Dissecting the conformational complexity and mechanism of a bacterial heme transporter

Dissecting the conformational complexity and mechanism of a bacterial heme transporter

Dissecting the conformational complexity and mechanism of a bacterial heme transporter

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

ヘム輸送体の立体構造と基質輸送メカニズム

ヘム輸送体の立体構造と基質輸送メカニズム

Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase

A heme-dependent conformational rearrangement of the C-terminal domain of heme binding protein (PhuS) is required for interaction with the iron-regulated heme oxygenase (HemO). Herein, we further investigate the underlying mechanism of this conformational rearrangement and its implications for heme transfer via site-directed mutagenesis, resonance Raman (RR), hydrogen-deuterium exchange MS (HDX-MS) methods, and molecular dynamics (MD). HDX-MS revealed that the apo-PhuS C-terminal α6/α7/α8-helices are largely unstructured, whereas the apo-PhuS H212R variant showed an increase in structure within these regions. The increased rate of heme association with apo-PhuS H212R compared with the WT and lack of a detectable five-coordinate high-spin (5cHS) heme intermediate are consistent with a more folded and less dynamic C-terminal domain. HDX-MS and MD of holo-PhuS indicate an overall reduction in molecular flexibility throughout the protein, with significant structural rearrangement and protection of the heme binding pocket. We observed slow cooperative unfolding/folding events within the C-terminal helices of holo-PhuS and the N-terminal α1/α2-helices that are dampened or eliminated in the holo-PhuS H212R variant. Chemical cross-linking and MALDI-TOF MS mapped these same regions to the PhuS:HemO protein-protein interface. We previously proposed that the protein-protein interaction induces conformational rearrangement, promoting a ligand switch from His-209 to His-212 and triggering heme release to HemO. The reduced conformational freedom of holo-PhuS H212R combined with the increase in entropy and decrease in heme transfer on interaction with HemO further support this model. This study provides significant insight into the role of protein dynamics in heme binding and release in bacterial heme transport proteins.

Direct Heme Uptake by Phytoplankton-Associated Roseobacter Bacteria

Iron is an essential micronutrient and can limit the growth of both marine phytoplankton and heterotrophic bacterioplankton. In this study, we investigated the molecular basis of heme transport, an organic iron acquisition pathway, in phytoplankton-associated Roseobacter bacteria and explored the potential role of bacterial heme uptake in the marine environment. We searched 153 Roseobacter genomes and found that nearly half contained putative complete heme transport systems with nearly the same synteny. We also examined a publicly available coculture transcriptome and found that Roseobacter strain Sulfitobacter sp. strain SA11 strongly downregulated a putative heme transport gene cluster during mutualistic growth with a marine diatom, suggesting that the regulation of heme transport might be influenced by host cues. We generated a mutant of phytoplankton-associated Roseobacter strain Ruegeria sp. strain TM1040 by insertionally inactivating its homolog of the TonB-dependent heme transporter hmuR and confirmed the role of this gene in the uptake of heme and hemoproteins. We performed competition experiments between iron-limited wild-type and mutant TM1040 strains and found that the wild type maintains a growth advantage when competing with the mutant for iron compounds derived solely from lysed diatom cells. Heme transport systems were largely absent from public marine metagenomes and metatranscriptomes, suggesting that marine bacteria with the potential for heme transport likely have small standing populations in the free-living bacterioplankton. Heme transport is likely a useful strategy for phytoplankton-associated bacteria because it provides direct access to components of the host intracellular iron pool after lysis. IMPORTANCE Ecosystem productivity in large regions of the surface ocean is fueled by iron that has been microbially regenerated from biomass. Currently, the specific microbes and molecules that mediate the transfer of recycled iron between microbial trophic levels remain largely unknown. We characterized a marine bacterial heme transporter and verified its role in acquiring heme, an abundant iron-containing enzyme cofactor. We present evidence that after host cell lysis, phytoplankton-associated bacteria directly extract heme and hemoproteins from algal cellular debris in order to fulfill their iron requirements and that the regulation of this process may be modulated by host cues. Direct heme transport, in contrast to multistep extracellular processing of hemoproteins, may allow certain phytoplankton-associated bacteria to rapidly extract iron from decaying phytoplankton, thus efficiently recycling cellular iron into the wider microbial loop.

Transmembrane Signaling through a Bacterial Heme Transporter

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of some nutrients is sensed by their specific outer membrane transporter. A cascade of molecular interactions between several proteins, located in three subcellular compartments, is then used to send this signal from outside to inside the bacteria and upregulate the expression of genes related to the acquisition of these nutrients. We study a heme acquisition system (Has) developed by several commensal and pathogenic bacteria to acquire heme as iron source. Using multidisciplinary approach (NMR, Xray, SAXS and Electron Microscopy) we have determined the structure of multiprotein complexes involved in the Has signaling pathway. Furthermore, we have recently shown, for the first time, that a partially folded protein is involved in this process 1,2,3,4,5. Our current data represent the first detailed characterization of this type of bacterial signaling. Ref: 1:Krieg S et al 2009 PNAS; 2:Caillet-Saguy S et al JACS 2009; 4: Cardoso de Amorim et al PlosOne 2013; Malki et al PlosOne 2014.Wojtowicz et al, in preparation.

Inward-facing conformation of the bacterial heme transporter

Iron is an essential element for almost all organisms, since iron serves as a catalytic center for redox reactions in many enzymes. Bacterial pathogens need to acquire iron from tissues of host to survive. Heme transport by ATP-binding cassette (ABC) transporter plays a key role in pathological processes. In gram-negative bacteria, the heme or heme protein binds to specific outer membrane receptors on the bacterial surface. The heme is then transported into the cell via ABC transporters. Here, we present the crystal structure of the heme transporter complex BhuUV-T from Burkholderia cenocepacia at 3.5 Å resolution in nucleotide-free state. The permeation pathway created by transmembrane helices of two BhuU subunit exhibits an inward-facing conformation. Comparison with the outward-facing conformation previously reported for the heme transporter HmuUV from Yersinia pestis and homologous vitamin B12 transporter BtuCD-F from E. coli indicates the structural mechanism involving the translational shift of nucleotide binding subunit and repositioning of the helices of permease subunits for substrate translocation. Structure of interface between BhuUV and periplasmic heme-binding protein BhuT suggests that the acidic residues of BhuU at the periplasmic interface may have an important role in releasing the heme from BhuT. We also determined the BhuT in apo and two types of holo form, providing the structural basis for transient and ambiguous heme recognition.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Metal-Substituted Protein MRI Contrast Agents Engineered for Enhanced Relaxivity and Ligand Sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.

Trafficking of heme and porphyrins in metazoa.

Trafficking of heme and porphyrins in metazoa.

Bacterial heme-transport proteins and their heme-coordination modes

Efficient iron acquisition is critical for an invading microbe’s survival and virulence. Most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. Utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. Once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in order to release the iron. Some Gram-negative bacteria also secrete extracellular heme-binding proteins called hemophores, which function to sequester heme from the environment. The heme-transport genes are often genetically linked as gene clusters under Fur (ferric uptake regulator) regulation. This review discusses the gene clusters and proteins involved in bacterial heme acquisition, transport and processing processes, with special focus on the heme-coordination, protein structures and mechanisms underlying heme-transport.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

Cofacial Heme Binding is Linked to Dimerization by a Bacterial Heme Transport Protein

Campylobacter jejuni is a leading bacterial cause of food-borne illness in the developed world. Like most pathogens, C. jejuni requires iron that must be acquired from the host environment. Although the iron preference of the food-borne pathogen C. jejuni is not established, this organism possesses heme transport systems to acquire iron. ChaN is an iron-regulated lipoprotein from C. jejuni proposed to be associated with ChaR, an outer-membrane receptor. Mutation of PhuW, a ChaN orthologue in Pseudomonas aeruginosa, compromises growth on heme as a sole iron source. The crystal structure of ChaN, determined to 1.9 Å resolution reveals that ChaN is comprised of a large parallel β-sheet with flanking α-helices and a smaller domain consisting of α-helices. Unexpectedly, two cofacial heme groups (∼3.5 Å apart with an inter-iron distance of 4.4 Å) bind in a pocket formed by a dimer of ChaN monomers. Each heme iron is coordinated by a single tyrosine from one monomer, and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Sequence analyses reveal that these residues are conserved among ChaN homologues from diverse bacterial origins. Electronic absorption and electron paramagnetic resonance (EPR) spectroscopy are consistent with heme binding through tyrosine coordination by ChaN in solution yielding a high-spin heme iron structure in a pH-dependent equilibrium with a low-spin species. Analytical ultracentrifugation demonstrates that apo-ChaN is predominantly monomeric and that dimerization occurs with heme binding such that the stability constant for dimer formation increases by 60-fold.