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

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

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

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

Arrangement of Annexin A2 tetramer and its impact on the structure and diffusivity of supported lipid bilayers

Annexins are a family of proteins that bind to anionic phospholipid membranes in a Ca2+-dependent manner. Annexin A2 forms heterotetramers (Anx A2t) with the S100A10 (p11) protein dimer. The tetramer is capable of bridging phospholipid membranes and it has been suggested to play a role in Ca2+-dependent exocytosis and cell-cell adhesion of metastatic cells. Here, we employ x-ray reflectivity measurements to resolve the conformation of Anx A2t upon Ca2+-dependent binding to single supported lipid bilayers (SLBs) composed of different mixtures of anionic (POPS) and neutral (POPC) phospholipids. Based on our results we propose that Anx A2t binds in a side-by-side configuration, i.e., both Anx A2 monomers bind to the bilayer with the p11 dimer positioned on top. Furthermore, we observe a strong decrease of lipid mobility upon binding of Anx A2t to SLBs with varying POPS content. X-ray reflectivity measurements indicate that binding of Anx A2t also increases the density of the SLB. Interestingly, in the protein-facing leaflet of the SLB the lipid density is higher than in the substrate-facing leaflet. This asymmetric densification of the lipid bilayer by Anx A2t and Ca2+ might have important implications for the biochemical mechanism of Anx A2t-induced endo- and exocytosis.Comment: 27 pages, 7 figures; supplementary material available upon request from the author

Changes in transcript and protein levels of calbindin D28k, calretinin and parvalbumin, and numbers of neuronal populations expressing these proteins in an ischemia model of rat retina

Excessive calcium is thought to be a critical step in various neurodegenerative processes including ischemia. Calbindin D28k (CB), calretinin (CR), and parvalbumin (PV), members of the EF-hand calcium-binding protein family, are thought to play a neuroprotective role in various pathologic conditions by serving as a buffer against excessive calcium. The expression of CB, PV and CR in the ischemic rat retina induced by increasing intraocular pressure was investigated at the transcript and protein levels, by means of the quantitative real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The transcript and protein levels of CB, which is strongly expressed in the horizontal cells in both normal and affected retinas, were not changed significantly and the number of CB-expressing horizontal cells remained unchanged throughout the experimental period 8 weeks after ischemia/reperfusion injury. At both the transcript and protein levels, however, CR, which is strongly expressed in several types of amacrine, ganglion, and displaced amacrine cells in both normal and affected retinas, was decreased. CR-expressing ganglion cell number was particularly decreased in ischemic retinas. Similar to the CR, PV transcript and protein levels, and PV-expressing AII amacrine cell number were decreased. Interestingly, in ischemic retinas PV was transiently expressed in putative cone bipolar cell types possibly those that connect with AII amacrine cells via gap junctions. These results suggest that these three calcium binding proteins may play different neuroprotective roles in ischemic insult by their ability to buffer calcium in the rat retina

Predicting small ligand binding sites in proteins using backbone structure

Motivation: Specific non-covalent binding of metal ions and ligands, such as nucleotides and cofactors, is essential for the function of many proteins. Computational methods are useful for predicting the location of such binding sites when experimental information is lacking. Methods that use structural information, when available, are particularly promising since they can potentially identify non-contiguous binding motifs that cannot be found using only the amino acid sequence. Furthermore, a prediction method that can utilize low-resolution models is advantageous because high-resolution structures are available for only a relatively small fraction of proteins

A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait

Genes that enable crops to limit Na+ accumulation in shoot tissues represent potential sources of salinity tolerance for breeding. In barley, the HvNax4 locus lowered shoot Na+ content by between 12% and 59% (g−1 DW), or not at all, depending on the growth conditions in hydroponics and a range of soil types, indicating a strong influence of environment on expression. HvNax4 was fine-mapped on the long arm of barley chromosome 1H. Corresponding intervals of ∼200 kb, containing a total of 34 predicted genes, were defined in the sequenced rice and Brachypodium genomes. HvCBL4, a close barley homologue of the SOS3 salinity tolerance gene of Arabidopsis, co-segregated with HvNax4. No difference in HvCBL4 mRNA expression was detected between the mapping parents. However, genomic and cDNA sequences of the HvCBL4 alleles were obtained, revealing a single Ala111Thr amino acid substitution difference in the encoded proteins. The known crystal structure of SOS3 was used as a template to obtain molecular models of the barley proteins, resulting in structures very similar to that of SOS3. The position in SOS3 corresponding to the barley substitution does not participate directly in Ca2+ binding, post-translational modifications or interaction with the SOS2 signalling partner. However, Thr111 but not Ala111 forms a predicted hydrogen bond with a neighbouring α-helix, which has potential implications for the overall structure and function of the barley protein. HvCBL4 therefore represents a candidate for HvNax4 that warrants further investigation

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