Annexins: a novel family of calcium- and membrane-binding proteins in search of a function

AbstractAlthough the annexins have been extensively studied and much detailed structural information is available, their in vivo function has yet to be established

The use of high halide-ion concentrations and automated phasing procedures for the structural analysis of BclA, the major component of the exosporium of Bacillus anthracis spores.

International audienceThe structure determination of the recombinant form of BclA, the major protein component of Bacillus anthracis exosporium, involved soaking in a high concentration of potassium iodide as the means of obtaining a good-quality heavy-atom derivative. The data to 2 angstroms resolution collected on a laboratory source were of sufficient quality to allow successful phasing and chain tracing by automated methods

Var2CSA Minimal CSA Binding Region Is Located within the N-Terminal Region

Var2CSA, a key molecule linked with pregnancy-associated malaria (PAM), causes sequestration of Plasmodium falciparum infected erythrocytes (PEs) in the placenta by adhesion to chondroitin sulfate A (CSA). Var2CSA possesses a 300 kDa extracellular region composed of six Duffy-binding like (DBL) domains and a cysteine-rich interdomain region (CIDRpam) module. Although initial studies implicated several individual var2CSA DBL domains as important for adhesion of PEs to CSA, new studies revealed that these individual domains lack both the affinity and specificity displayed by the full-length extracellular region. Indeed, recent evidence suggests the presence of a single CSA-binding site formed by a higher-order domain organization rather than several independent binding sites located on the different domains. Here, we search for the minimal binding region within var2CSA that maintains high affinity and specificity for CSA binding, a characteristic feature of the full-length extracellular region. Accordingly, truncated recombinant var2CSA proteins comprising different domain combinations were expressed and their binding characteristics assessed against different sulfated glycosaminoglycans (GAGs). Our results indicate that the smallest region within var2CSA with similar binding properties to those of the full-length var2CSA is DBL1X-3X. We also demonstrate that inhibitory antibodies raised in rabbit against the full-length DBL1X-6ε target principally DBL3X and, to a lesser extent, DBL5ε. Taken together, our results indicate that efforts should focus on the DBL1X-3X region for developing vaccine and therapeutic strategies aimed at combating PAM

Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain III

AbstractThe domain III of annexin 5 undergoes a Ca2+- and a pH-dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID–IIIE loop on the one hand and the H-bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065–14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH- and Ca2+-mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal

Allelic Diversity of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Entails Variant-Specific Red Cell Surface Epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α1 PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α1 domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquistion of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity

Détermination de la structure tridimensionnelle de la protéine YodA de E. Coli afin de proposer une fonction

YodA de E. coli est une protéine de stress, obtenue en quantité importante lors de la sur-expression en fermenteur d'une souche E.coli contenant un plasmide codant pour la transacylase CyaC. La similarité de comportement de YodA et CyaC dans la caractérisation biochimique était forte et seule la spectrométrie de masse a révélé sans ambiguïté que la protéine étudiée était YodA, protéine endogène de stress. La détermination de sa structure tridimensionnelle par diffraction des rayons X a été réalisée sur trois formes cristallines obtenues en absence et en présence de cations: le zinc et le cadmium. Ces structures ont été affinées à des résolutions proches de 2.0 A, suffisantes pour interpréter l'information structurale. YodA est formée de deux domaines structuraux mais ne présente pas de similarité globale de structure. On peut la considérer comme modèle d'un repliement nouveau, représentatif d'une sous-famille de protéines de séquences similaires à YodA (YrpE de B. subtilis et pXO1-130 de B. anthracis). Le nombre et la position de ces cations varient d'une structure à l'autre: l'addition de zinc conduit à trois sites de fixations, celle de cadmium à quatre sites. La protéine dite native fixe un ion nickel, sans doute libéré de la colonne d'affinité utilisée lors de la purification. Cet évènement illustre la tendance de la protéine à fixer des cations dans un site majeur, occupé quelque soit la structure. Ce site implique trois résidus histidines et l'hypothèse est ainsi faite qu'une des fonctions biologiques de YodA serait la fixation et le transport de cations. Ce lien est possible dans la mesure où il est connu que l'apparition de YodA dans le périplasme de E. coli est associée au stress crée par la présence de cadmium dans le milieu intracellulaire. YodA apparaît également en présence d'un autre agent de stress, le peroxyde d'hydrogène. Cette étude peut être une base de recherches futures sur YodA comme intervenant dans le processus de réponses au stress.YodA of E.coli is a protein of stress, obtained in significant quantity during the over-expression in fermentor of E.coli strain containing a plasmid carling for the transacylase CyaC. The similarity of behaviour of YodA and CyaC in the biochemical characterization was strong and only the mass spectrometry revealed without ambiguity that the isolated protein was YodA, an endogenous protein of stress. The determination of its three-dimensional structure by diffraction of x-rays was carried out on three crystal forms obtained in absence and in the presence of cations: zinc and cadmium. These structures were refined with resolutions close to 2.0 A, sufficient to interpret all the structural information's. YodA is made of two structural domains but does not present any relevant similarity with known structures. One can consider it as a model of a new folding, representative protein of a subfamily of sequences similar to YodA (YrpE of B subtilis and pXO1-130 of B anthracis). The number and the position of cations vary from a structure to another: zinc addition leads to three sites while cadmium complexes four sites. It is remarkable that the protein known as native also fixes a nickel, which undoubtedly is released from the affinity column used during the purification. This event illustrates the tendency of the protein to complex cations in a major site, always occupied in the structure. This site implies three histidine residues and the hypothesis is thus made that the biological functions of YodA would be the complexation and transport of cations. The validity of this hypothesis is corroborated as YodA is always associated with the stress induced by cadmium in the intracellular medium YodA also appears in the presence of hydrogen peroxide, another known agent of stress. This study may be the base for future research on YodA in the process of stress responses.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Statistical experimental design of protein crystallization screening revisited.

A statistical experimental design approach was used to prepare a set of solutions for the screening of protein crystallization conditions. This approach is shown to be amenable to quantitative evaluation and therefore to the rational optimization of the screening results. All solutions contain a cryoprotectant, thus eliminating the need for subsequent optimization of crystal freezing conditions

Investigation of molecular and protein crystals by three photon polarization resolved microscopy.

International audienceWe implement three photon fluorescence polarization resolved microscopy to optically investigate molecular and protein crystals. The availability of UV transitions using IR pulses allows analyses without fluorescence staining. Polarization resolved studies indicate that high-order symmetry structures can be revealed and that strong fluorescent energy transfer occurs between molecules. We show how this permits identification of a monocrystalline nature for a sample at the subwavelength resolution scale