Anita Lewit-Bentley

EF-hand calcium-binding proteins.

The EF-hand motif is the most common calcium-binding motif found in proteins. Several high-resolution structures containing different metal ions bound to EF-hand sites have given new insight into the modulation of their binding affinities. Recently determined structures of members of several newly identified protein families that contain the EF-hand motif in some of their domains, as well as of their complexes with target molecules, are throwing light on the surprising variety of functions that can be served by this simple and ingenious structural motif.

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11. p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties.We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

Structural Basis of the Ca2+ Dependent Association between S100C (S100A11) and its Target, the N-Terminal Part of Annexin I

BACKGROUND S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. RESULTS We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. CONCLUSIONS By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA

Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (KD = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.

Neutron diffraction studies on crystals of nucleosome cores using contrast variation

Abstract Neutron diffraction data have been collected from crystals of intact nucleosome core particles to a resolution of 25 A. By varying the proportion of D 2 O, the scattering of the mother liquor relative to the protein and DNA can be altered. At 39% D 2 O, the solvent scattering matches that of the protein and so only the DNA is scattering, and similarly at 65% D 2 0 only the protein scatters. Using this approach the neutron scattering of the two components and of the complete particle (0% D 2 O) have been measured. The data corresponding to the principal projections are consistent with a model in which 1.8 turns of a DNA superhelix of pitch 27·5 A and radius 42 A are wound around a protein core.

S100-annexin complexes: some insights from structural studies.

Several annexins have been shown to bind proteins that belong to the S100 calcium‐binding protein family. The two best‐characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N‐terminus and of S100C with annexin I N‐terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42–55), we propose a computer generated model for the entire annexin II/p11 complex.

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

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

Crystal structure of the nucleosome core particle at 16 Å resolution

The crystal structure of the nucleosome core particle has been studied by neutron diffraction to a resolution of 16 A. By using H2O/D2O solvent contrast variation, the structures of the DNA and histone core were analysed separately. The DNA, as seen at this resolution, forms a super-helix of pitch 25.8 A, radius 42.1 A and 1.8 turns in length. The histone core itself is approximately helical and follows the DNA along the inside of the super-helix, giving the nucleosome core particle an overall 2-fold axis of symmetry. Four regions can be distinguished in the protein density, which we interpret as dimers of histones within the octameric core. The dimers have been assigned on the basis of other evidence as being of two kinds, (H2A-H2B) and (H3-H4). Because solvent contrast variation can distinguish between hydrophobic and hydrophilic regions in the protein density, our results suggest that the interface between the monomers of each dimer is probably quite hydrophobic in character, while the interaction between dimers is weaker and/or more hydrophilic. The protein is in contact with most of the DNA and there are some regions where it may penetrate between the turns of the super-helix. In particular, the tetramer (H4-H3)-(H3-H4) is in close contact with the central part of the DNA, but significant contacts are seen also between the histones H3 and the extremities of the super-helix, thus explaining the stability of a nucleosome-like particle depleted of H2A and H2B. Significant departures from the molecular 2-fold axis of symmetry occur in the relative arrangements of the two (H2A-H2B) dimers.

YodA from Escherichia coli is a Metal-binding, Lipocalin-like Protein

We have determined the crystal structure of YodA, an Escherichia coli protein of unknown function. YodA had been identified under conditions of cadmium stress, and we confirm that it binds metals such as cadmium and zinc. We have also found nickel bound in one of the crystal forms. YodA is composed of two domains: a main lipocalin/calycin-like domain and a helical domain. The principal metal-binding site lies on one side of the calycin domain, thus making YodA the first metal-binding lipocalin known. Our experiments suggest that YodA expression may be part of a more general stress response. From sequence analogy with the C-terminal domain of a metal-binding receptor of a member of bacterial ATP-binding cassette transporters, we propose a three-dimensional model for this receptor and suggest that YodA may have a receptor-type partner in E. coli.

Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition

The human malaria parasite Plasmodium falciparum can cause infected red blood cells (iRBC) to form rosettes with uninfected RBC, a phenotype associated with severe malaria. Rosetting is mediated by a subset of the Plasmodium falciparum membrane protein 1 (PfEMP1) variant adhesins expressed on the infected host-cell surface. Heparin and other sulfated oligosaccharides, however, can disrupt rosettes, suggesting that therapeutic approaches to this form of severe malaria are feasible. We present a structural and functional study of the N-terminal domain of PfEMP1 from the VarO variant comprising the N-terminal segment (NTS) and the first DBL domain (DBL1α1), which is directly implicated in rosetting. We demonstrate that NTS-DBL1α1-VarO binds to RBC and that heparin inhibits this interaction in a dose-dependent manner, thus mimicking heparin-mediated rosette disruption. We have determined the crystal structure of NTS-DBL1α1, showing that NTS, previously thought to be a structurally independent component of PfEMP1, forms an integral part of the DBL1α domain. Using mutagenesis and docking studies, we have located the heparin-binding site, which includes NTS. NTS, unique to the DBL α-class domain, is thus an intrinsic structural and functional component of the N-terminal VarO domain. The specific interaction observed with heparin opens the way for developing antirosetting therapeutic strategies.