Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design.

The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunization with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+ T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid.

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

A route to self-assemble suspended DNA nano-complexes.

Highly charged polyelectrolytes can self-assemble in presence of condensing agents such as multivalent cations, amphiphilic molecules or proteins of opposite charge. Aside precipitation, the formation of soluble micro- and nano-particles has been reported in multiple systems. However a precise control of experimental conditions needed to achieve the desired structures has been so far hampered by the extreme sensitivity of the samples to formulation pathways. Herein we combine experiments and molecular modelling to investigate the detailed microscopic dynamics and the structure of self-assembled hexagonal bundles made of short dsDNA fragments complexed with small basic proteins. We suggest that inhomogeneous mixing conditions are required to form and stabilize charged self-assembled nano-aggregates in large excess of DNA. Our results should help re-interpreting puzzling behaviors reported for a large class of strongly charged polyelectrolyte systems.

Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone.

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970-1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263-1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.

Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

H2A and H2B tails are essential to properly reconstitute nucleosome core particles.

The conformation of recombinant Nucleosome Core Particles (NCPs) lacking H2A and H2B histone tails (gH2AgH2B) are studied. The migration of these particles in acrylamide native gels is slowed down compared to intact reconstituted NCPs. gH2AgH2B NCPs are also much more sensitive to nuclease digestion than intact NCPs. Small angle X-ray scattering (SAXS) experiments point out that the absence of H2A and H2B tails produces small but significant conformational changes of the octamers conformation (without wrapped DNA), whereas gH2AgH2B NCP conformations are significantly altered. A separation of about 25-30 bp from the core could account for the experimental curves, but other types of DNA superhelix deformation cannot be excluded. The distorted gH2AgH2B octamer may not allow the correct winding of DNA around the core. The absence of the H2A and H2B tails would further prevent the secondary sliding of the DNA around the core and therefore impedes the stabilisation of the particle. Cryo-electron microscopy on the same particles also shows a detachment of DNA portions from the particle core. The effect is even stronger because the vitrification of the samples worsens the instability of gH2AgH2B NCPs.

Structure and phase diagram of nucleosome core particles aggregated by multivalent cations.

The degree of compaction of the eukaryotic chromatin in vivo and in vitro is highly sensitive to the ionic environment. We address the question of the effect of multivalent ions on the interactions and mutual organization of the chromatin structural units, the nucleosome core particles (NCPs). Conditions of precipitation of NCPs in the presence of 10 mM Tris buffer and various amounts of either magnesium (Mg(2+)) or spermidine (Spd(3+)) are explored, compared, and discussed in relation to theoretical models. In addition, the structure of the aggregates is analyzed by complementary techniques: freeze-fracture electron microscopy, cryoelectron microscopy, and x-ray diffraction. In Mg(2+)-NCP aggregates, NCPs tend to stack on top of one another to form columns that are not long-range organized. In the presence of Spd(3+), NCPs precipitate to form a dense isotropic phase, a disordered phase of columns, a two-dimensional columnar hexagonal phase, or a three-dimensional crystal. The more ordered phases (two-dimensional or three-dimensional hexagonal) are found close to the precipitation line, where the number of positive charges carried by cations is slightly larger than the number of available negative charges of the NCPs. All ordered phases coexist with the dense isotropic phases. Formation of hexagonal and columnar phases is prevented by an excess of polycations.

H3 and H4 histone tails play a central role in the interactions of recombinant NCPs.

Using small-angle x-ray scattering, we probe the effect of histone tails on both internucleosomal interactions and nucleosome conformation. To get insight into the specific role of H3 and H4 histone tails, perfectly monodisperse recombinant nucleosome core particles were reconstituted, either intact or deprived of both H3 and H4 histone tails (gH3gH4). The main result is that H3 and H4 histone tails are necessary to induce attractive interactions between NCPs. A pair potential model was used to describe interactions between NCPs. At all salt concentrations, interactions between gH3gH4 NCPs are best described by repulsive interactions exclusively. For intact NCPs, an additional attractive term, with a 5-10 kT magnitude and 20 A range, is required to account for interparticle interactions above 50 mM monovalent salt. Regarding conformation, intact NCPs in solution are similar to NCPs in 3D crystals. gH3gH4 NCPs instead give rise to slightly different small-angle x-ray scattering curves that can be understood as a more opened conformation of the particle, where DNA ends are slightly detached from the core.

The crystal structure of annexin A8 is similar to that of annexin A3.

Annexin A8 is a relatively infrequent and poorly studied member of this large family of calcium-binding and membrane-binding proteins. It is, however, associated with a specific disease, acute promyelocytic leukemia. We have solved its three-dimensional structure, which includes a moderately long and intact N terminus. The structure is closest to that of annexin A3 and highlights several important regions of inherent flexibility in the annexin molecule. The N terminus resembles that of annexin A3, as it lies along the concave surface of the molecule and inserts partially into the hydrophilic channel in its centre. Since both annexins A3 and A8 are expressed in promyelocytic cells during their differentiation, the similarity in their structures might suggest a functional relationship.

Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy.

The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, Ant-dATP) and a cysteine-specific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC-CaM, similar to that observed for isolated CaM-binding sequences of different lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Bârzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem.237, 619-628]. In contrast, Ant-dATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein's hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of approximately 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60-70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of approximately 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach (hydropro), which uses the 3D structure [Drum, C.L., Yan, S.-Z., Bard, J., Shen, Y.-Q., Lu, D., Soelalman, S., Grabarek, Z., Bohm, A. & Tang, W.-J. (2002) Nature (London)415, 396-402]. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different.

Crystallization and preliminary analysis of Escherichia coli YodA.

The Escherichia coli protein YodA was overexpressed, purified and crystallized in several crystal forms. The function of this protein is not known, although it has been identified under conditions of bacterial stress. Three of the four crystal forms were obtained in the presence of divalent cations (zinc, nickel and cadmium), suggesting that YodA may be a metal-binding protein.