Struktur und Funktion des Aussenmembranrezeptors HasR aus Serratia marcescens im Komplex mit dem Hämophorprotein HasA

Dans la plupart des cas, l entrée des sources de fer dans les bactéries à Gram-négatif, dépend d un transport actif à travers la membrane externe. Des récepteurs spécifiques de membrane externe reconnaissent les substrats à transporter ; sous l action de la force proton-motrice de la membrane interne, « transduite » par un complexe de membrane interne (le complexe TonB-ExbB-ExbD) au récepteur, le substrat est transféré dans le périplasme. Parmi les différentes sources de fer disponibles, la bactérie à Gram-négatif Serratia marcescens est capable d utiliser l hème extracellulaire, par son système spécifique Has (Haem Acquisition System). Les composants essentiels de ce système sont un récepteur spécifique de membrane externe HasR, une protéine monomérique sécrétée, HasA, appelée hémophore ayant une très grande affinité pour l hème (Ka = 5.3 × 1010 M-1), et une protéine, HasB, homologue à TonB. HasA reconnaît spécifiquement le récepteur HasR et sa présence augmente l efficacité du système à acquérir l hème à de très faibles concentrations. Le transport de l hème par HasR, à travers la membrane externe, dépend d un complexe de membrane interne TonB(HasB)-ExbB-ExbD, utilisant l énergie de la force proton-motrice pour l internalisation de l hème, et le recyclage de l hémophore HasA dans le milieu extracellulaire. Ce système est reconstitué de manière fonctionnelle, chez Escherichia coli.Les protéines HasA et HasR ont été purifiées en présence et en absence d hème ; leurs caractéristiques spectrales et leurs interactions ont été déterminées. L affinité entre HasA et HasR est élevée (1010 M-1) et un complexe stable de stoechiométrie 1 : 1 peut être formé in vitro, indépendamment de la présence d une molécule d hème. HasR fixe une molécule d hème avec une affinité plus faible que celle de HasA (Ka = 5 × 106 M-1). Les caractéristiques spectrales (UV-visible et Raman) de HoloHasR sont identiques à celles du complexe établi entre HoloHasA et ApoHasR, et différentes de celles de HoloHasA. De ces résultats, nous proposons que l interaction entre HoloHasA et ApoHasR permette un transfert de l hème du site de fixation de HasA sur le site de fixation de HasR, sans apport d énergie. Le site de fixation de l hème sur le récepteur implique vraisemblablement deux histidines conservées chez les récepteurs à hème. La mutation de ces résidus en alanine abroge l activité de transport d hème du récepteur sans empêcher l interaction hémophore/récepteur.L alignement de séquence et la prédiction de structure secondaire montre une homologie de structure avec certains récepteurs aux sidérophores comme FecA dont la structure tridimensionnelle existe. La structure de HoloHasA est connue, mais comme aucune structure de récepteur à hème n est encore disponible, nous avons voulu résoudre la structure cristalline du complexe HoloHasA-HasR. HoloHasA est purifié dans une version fonctionnelle, étiquetée de six histidines en N-terminal. Le complexe HoloHis6-HasA-HasR cristallise dans le groupe d espace P212121. Les cristaux croissent dans des assemblages hétérogènes de plaques et d aiguilles de 0,01 à 0,1 × 0,1 × 1 mm. Un jeu natif de données cristallographiques a été collecté. La résolution atteinte est 6,8 Å bien que des réflexions jusqu à 4 Å soient observées. L anisotropie importante entre 6 et 4 Å ne permet pas la détermination des phases nécessaires à la détermination d une structure.

Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

The expression, purification, and crystallization in space group P212121 of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å

Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex.

International audienceGram-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

The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site.

International audienceHasA 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

Structuring detergents for extracting and stabilizing functional membrane proteins.

BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field

BmrA purification with C4Cn and detergent exchange.

<p>(<b>A</b>) SDS-PAGE of the sequential extraction of BmrA. As detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>, the membrane fraction (lane T) was incubated with C4C3 and then centrifuged to give the supernatant S and the pellet P. The latter, enriched in BmrA, was suspended in the presence of C4C7 and then centrifuged to give the corresponding supernatant S and pellet P. Arrows indicate the position of BmrA, C4C3 and C4C7. The C4C7 supernatant was then subjected to DLS (B), Ni-affinity chromatography (<b>C</b>) and gel filtration carried out with FC12 (<b>D</b>) from which respective pools indicated by stars were loaded onto SDS-PAGE.</p

Extraction of membrane proteins by C4Cn derivatives.

<p>Extraction of ABC transporters by C4Cn, prokaryotic BmrA (A) and YheI/YheH (B) expressed in <i>E. coli</i>, human ABCG2 expressed in <i>Sf</i>9 (C) or HEK293 (D) cells, together with AcrB expressed in <i>E. coli</i> (E) and the SR-Ca²⁺-ATPase (F), was carried out on the corresponding membrane fractions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. After solubilisation, extracted and non-extracted materials were separated by high-speed centrifugation, generating a supernatant S and a pellet P which were loaded on a 10% SDS-PAGE, and after migration either stained with Coomassie blue or submitted to a Western blot for ABCG2 (upper lanes in panels in C and D). Arrows indicate the position of each monomer. Positive control experiments were carried out with DDM, FC12 and C12E8, negative controls were carried out without detergent (<i>No det</i>). The red dotted line indicates the threshold of extraction.</p

Crystallization of BmrA extracted with C4C10.

<p>BmrA was extracted and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g005" target="_blank">Figure 5</a>, using C4C10 instead of C4C7 and exchanging it with FC12. (A) The protein, concentrated to 10 mg/ml, and mixed with 1 mM doxorubicin, crystallized after 10 days in 0.2 M KSCN, 20% PEG 3350, and was (B) analyzed at the ESRF beamline ID23EH-2.</p

C4Cn preserve a functional state of BmrA.

<p>(<b>A</b>) Binding of daunorubicin to BmrA monitored by intrinsic (Tryptophan, Trp) fluorescence quenching. BmrA was extracted and purified either with C4C10 (circles) or FC12 (triangles) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a>, the former being subsequently exchanged by FC12 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4D</a>. The purified protein was then incubated with increasing concentrations of daunorubicin, the binding of the drug being probed by the variation of intrinsic fluorescence of BmrA, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. (<b>B</b>) VO₄-sensitive ATPase activity of BmrA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>) in different fractions: BmrA-enriched membrane fraction (“-“ bar) corresponding to 0.5 µmol/min.mg and taken as 100%; BmrA-enriched membrane fraction solubilized with 1% SDS, FC12, DDM, or C4C3+C4C7 (“Extraction/C4Cn” bar, carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Fig. 4</a>); BmrA extracted with FC12 and then purified by metal affinity and gel filtration with FC12 (“Purification/FC12” bar); BmrA extracted with C4C3+C4C7 and then purified by metal affinity with C4C7 followed by detergent exchange with FC12 using gel filtration as carried out in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a> (“Purification/C4Cn, FC12 exchange” bar). (<b>C</b>) Intrinsic fluorescence quenching monitoring of C4C10 binding on BmrA. BmrA was extracted either with C4C10 or FC12 and then purified with FC12 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a> generating two populations on which C4C10 binding was monitored by probing the quenching of intrinsic fluorescence of 1 µM BmrA as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>.</p

C4Cn behaviour in solution and interaction with basic amino acids.

<p>(A) <i>Effects of increasing C4C1, C4C3, C4C7 and C4C12 concentrations on the surface tension of aqueous solution</i>. The surface tension is measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. Each value is the mean of three experiments ± the standard error. C4C1, C4C3, C4C7 and C4C12 are indicated by circles, diamonds, triangles and squares, respectively. (B) <i>Dynamic light scattering of C4Cn in aqueous solution</i>. Experiments were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. Mean size values are indicated in nm for the corresponding compound. (C) <i>Effect of pH on the surface tension generated by C4C7</i>. Experiments have been carried out as in (A), neutralizing C4C7 at pH 9.0, 8.0 and 6.0, and measuring the resulting surface tension by increasing the concentration of the compound, as indicated by circles, triangles and squares, respectively. The values result from triplicate experiments. (D) <i>Interaction of C4C7 with amino acids probed by surface tension</i>. C4C7 diluted to 10 µM was incubated with increasing concentrations of either glutamate (circles), lysine (triangles), arginine (squares) or proline (diamonds), measuring the resulting surface tension, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. (E) <i>Chemical shifts of the αH and εH protons of lysine in the presence of C4C1</i>. The ¹H NMR spectra of L-Lysine (10 mM) was recorded as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a> in the presence of increasing C4C1 concentrations as indicated in the Figure, resulting in chemical shifts of αH and εH protons of lysine which were plotted as a function of C4C1 concentration. (F). <i>Dissociation of the L-lysine - C4C1 complex</i>, each added at 10 mM, induced by increasing the salt concentration as indicated and probed by measuring the chemical shift of lysine αH protons.</p