Le transporteur mitochondrial de nucléotides adényliques de S. cerevisiae (état oligométrique et cristallogénèse tridimentionnelle)

Un lien énergétique essentiel entre le cytoplasme et la mitochondrie d'une cellule eucaryote est le transporteur ADP/ATP, ou Ancp, localisé dans la membrane mitochondriale interne. Différentes méthodes expérimentales ont permis de montrer que l'Ancp pourrait être actif sous forme de dimère. Cependant, la résolution de la structure de l'Ancp de boeuf inhibé par le carboxyatractyloside a révélé un monomère dans le cristal, remettant en question les données concernant son oligomérisation. De manière à retenir artificiellement l'hypothétique dimère durant la cristallisation, nous utilisons un dimère covalent en tandem d'Ancp de levure. Nous avons mis au point un protocole de purification pour ce dimère afin de permettre sa cristallisation et la résolution de sa structure. Ainsi, des données structurales sur cette protéine artificiellement dimérique permettront de répondre aux interrogations concernant l'oligomérisation d'Ancp.An essential energy connection between cytoplasm and mitochondria cells is the ADP/ATP carrier, or Ancp, located in the mitochondrial inner membrane. Various experimental methods have indicated that Ancp might fullfill its transport function in a dimeric state. However the determination of the structure of bovine Ancp reveals a monomer in the cristal, calling the data relating to its oligomerization into question. In order to artificially retain the hypothetical dimer during crystallization we are using a covalent tandem dinner of yeast Ancp. We have developed a purification for this dinner to enable its crystallization and resolution of its structure. This structural data on this artificial dimeric protein will answer questions about the oligomerization of Ancp.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Crystal Structure of the Vanadate-Inhibited Ca2+\mathrm{Ca^{2+}}-ATPase

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca$^{2+}$-ATPase with bound vanadate in the absence of Ca$^{2+}$. Vanadate is bound at the catalytic site as a planar VO$_3$− in complex with water and Mg$^{2+}$ in a dephosphorylation transition-state-like conformation. Validating bound VO$_3$− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca$^{2+}$-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO$_3$− structure with that in E2·BeF$_3$− (E2P ground state analog) reveals multiple binding modes to the Ca$^{2+}$-ATPase

Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast

International audienceWe isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region