RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Hemo/metabolismo , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Serratia marcescens/metabolismo , Espectrometría RamanRESUMEN
Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 A from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Membrana Celular/metabolismo , Hemo/metabolismo , Hemoproteínas/química , Proteínas de la Membrana/química , Receptores de Superficie Celular/química , Serratia marcescens/química , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Calorimetría , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Hemo/química , Proteínas de Unión al Hemo , Hemoproteínas/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Secundaria de Proteína , Receptores de Superficie Celular/metabolismo , Propiedades de SuperficieRESUMEN
The first step of heme acquisition by Gram-negative pathogenic bacteria through the so-called heme acquisition system, Has, requires delivery of the heme from the extracellular hemophore protein HasA to a specific outer membrane receptor, HasR. CRINEPT-TROSY NMR experiments in DPC micelles were here used to obtain information on the intermediate HasA-HasR complex in solution. A stable protein-protein adduct is detected both in the presence and in the absence of heme. Structural information on the complexed form of HasA is obtained from chemical shift mapping and statistical analysis of the spectral fingerprint of the protein NMR spectra obtained under different conditions. This approach shows the following: (i) only three different conformations are possible for HasA in solution: one for the isolated apoprotein, one for the isolated holoprotein, and one for the complexed protein, that is independent of the presence of the heme; (ii) the structure of the hemophore in the complex resembles the open conformation of the apoprotein; (iii) the surface contact area between HasA and HasR is independent of the presence of the heme, involving loop L1, loop L2, and the beta2-beta6 strands; (iv) upon complex formation the heme group is transferred from holoHasA to HasR.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Hemo/química , Hemo/metabolismo , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Conformación ProteicaRESUMEN
To satisfy their iron needs, several Gram-negative bacteria use a heme uptake system involving an extracellular heme-binding protein called hemophore. The function of the hemophore is to acquire free or hemoprotein-bound heme and to transfer it to HasR, its specific outer membrane receptor, by protein-protein interaction. The hemophore HasA secreted by Serratia marcescens, an opportunistic pathogen, was the first to be identified and is now very well characterized. HasA is a monomer that binds one b heme with strong affinity. The heme in HasA is highly exposed to solvent and coordinated by an unusual pair of ligands, a histidine and a tyrosine. Here, we report the identification, the characterization and the X-ray structure of a dimeric form of HasA from S. marcescens: DHasA. We show that both monomeric and dimeric forms are secreted in iron deficient conditions by S. marcescens. The crystal structure of DHasA reveals that it is a domain swapped dimer. The overall structure of each monomeric subunit of DHasA is very similar to that of HasA but formed by parts coming from the two different polypeptide chains, involving one of the heme ligands. Consequently DHasA binds two heme molecules by residues coming from both polypeptide chains. We show here that, while DHasA can bind two heme molecules, it is not able to deliver them to the receptor HasR. However, DHasA can efficiently transfer its heme to the monomeric form that, in turn, delivers it to HasR. We assume that DHasA can function as a heme reservoir in the hemophore system.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X/métodos , Proteínas de la Membrana/metabolismo , Serratia marcescens/metabolismo , Proteínas Bacterianas/metabolismo , Dimerización , Escherichia coli/metabolismo , Hemo/química , Hemina/química , Histidina/química , Ligandos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Tirosina/químicaRESUMEN
The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras , Galio/metabolismo , Hemo/metabolismo , Histidina/química , Proteínas de la Membrana/química , Protoporfirinas/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.
Asunto(s)
Monóxido de Carbono/metabolismo , Hemo/metabolismo , Mutación , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Sondas Moleculares , Oxidación-Reducción , Espectrometría RamanRESUMEN
A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity. Its alpha/beta structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore beta-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 A, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Serratia marcescens/química , Hemo/metabolismo , Proteínas de Unión al Hemo , Hemoproteínas , Ligandos , Luz , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de RadiaciónRESUMEN
Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Hemo/química , Histidina/química , Hierro/química , Proteínas de la Membrana/química , Serratia marcescens/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hemo/genética , Hemo/metabolismo , Histidina/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Hierro/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación Missense , Unión Proteica , Estructura Terciaria de Proteína , Serratia marcescens/genética , Serratia marcescens/metabolismo , Relación Estructura-ActividadRESUMEN
Hemophore HasA is a 19 kDa iron(III) hemoprotein that participates in the shuttling of heme to a specific membrane receptor. In HasA, heme iron has an original coordination environment with a His/Tyr pair as axial ligands. Recently developed two-dimensional protonless (13)C-detected experiments provide the sequence-specific assignment of all but three protein residues in the close proximity of the paramagnetic center, thus overcoming limitations due to the short relaxation times induced by the presence of the iron(III) center. Mono-dimensional (13)C and (15)N experiments tailored for the detection of paramagnetic signals allow the identification of resonances of the axial ligands. These experiments are used to characterize the conformational features and the electronic structure of the heme iron(III) environment. The good complementarity among (1)H-, (13)C-, and (15)N-detected experiments is highlighted. A thermal high-spin/low-spin equilibrium is observed and is related to a modulation of the strength of the coordination bond between the iron and the Tyr74 axial ligand. The key role of a neighboring residue, His82, for the stability of the axial coordination and its involvement in the heme delivery to the receptor is discussed.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Hemoproteínas/química , Proteínas de la Membrana/química , Animales , Anisotropía , Sitios de Unión , Isótopos de Carbono , Bovinos , Hemo/química , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 x 10(10) m(-1)). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 x 10(6) m(-1) and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA.HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/fisiología , Sitios de Unión , Calorimetría , Proteínas Portadoras/química , Cinética , Proteínas de la Membrana/química , Mutación , Plásmidos/metabolismo , Unión Proteica , Receptores de Superficie Celular/metabolismo , Espectrofotometría , Espectrometría Raman , Termodinámica , Rayos UltravioletaRESUMEN
HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.