Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

Ultrastructural organization of dentin in mice lacking dentin sialo-phosphoprotein.

Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.

Synthesis of bone-like nanocomposites using multiphosphorylated peptides.

There is a great need for novel materials for mineralized tissue repair and regeneration. Two examples of such tissue, bone and dentin, are highly organized hierarchical nanocomposites in which mineral and organic phases interface at the molecular level. In contrast, current graft materials are either ceramic powders or physical blends of mineral and organic phases with mechanical properties far inferior to those of their target tissues. The objective of this study was to synthesize composite nanofibrils with highly integrated organic/inorganic phases inspired by the mineralized collagen fibrils of bone and dentin. Utilizing our understanding of bone and dentin biomineralization, we have first designed bioinspired peptides containing 3 Ser-Ser-Asp repeat motifs based on the highly phosphorylated protein, dentin phosphophoryn (DPP), found in dentin and alveolar bone. We demonstrate that up to 80% of serines in the peptide can be phosphorylated by casein kinases. We further tested the ability of these peptides to induce biomimetic calcium phosphate mineralization of collagen fibrils. Our mineralization studies have revealed that in the presence of these phosphorylated peptides, mineralized collagen fibrils structurally similar to the mineralized collagen fibrils of bone and dentin were formed. Our results demonstrate that using phosphorylated DPP-inspired peptides, we can successfully synthesize biomimetic composite nanofibrils with integrated organic and inorganic phases. These results provide the first step in the development of biomimetic nanostructured materials for mineralized tissue repair and regeneration using phosphopeptides.

CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro.

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale.

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization.

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.

Cryogenic transmission electron microscopy study of amelogenin self-assembly at different pH.

Cryogenic transmission electron microscopy (cryo-EM) was used to explore the self-assembly of recombinant murine amelogenin (rM179) in vitro. Our cryo-EM data showed that amelogenin self-assembly is a strongly pH-dependent process. At pH 4.4 the main fraction of the protein exists in a monomeric form, although some peculiar structures consisting of chains of monomers were also observed. At pH 5.8 large nanospheres comprising ring-like structures ~50 nm in diameter were the most abundant particle class. Similarly, at pH 8.0 amelogenins self-assembled into ring-like oligomers of different sizes, which subsequently assembled into nanospheres 15-20 nm in diameter. Furthermore, at pH 7.2, which is close to a physiological pH, branched chains of nanospheres were observed. Our results show that amelogenin assembly is a multistep hierarchical process and provides new insight into the control of enamel mineralization.

Terminal modifications of norovirus P domain resulted in a new type of subviral particles, the small P particles.

The protruding (P) domain of norovirus VP1 is responsible for immune recognition and host receptor interaction. Our previous studies have demonstrated that a modification of the ends of the P domain affects the conformation and/or function of the P protein. An expression of the P domain with or without the hinge, or with an additional cysteine at either ends of the P protein resulted in P dimers and/or P particles. Here we report a new type of subviral particle, the small P particles, through a further modification, either an addition of the flag tag or a change of the arginine cluster, at the C-terminus of the cysteine-containing P domain. Gel filtration and cryo-EM studies showed that the small P particles are tetrahedrons formed by 6 P dimers or 12 P monomers that is half-size of the P particles. Fitting of the crystal structure of the P domain into the cryo-EM density map of the particle indicated similar conformations of the P dimers as those in P particles. The small P particles bind human HBGAs and are antigenically reactive similar to their parental VLPs and P particles. These data suggest that the C-terminus of the P domain is an important factor in the formation of the P particles. Further elucidation of the mechanism of these modifications in the P particle formation would be important in structure biology and morphogenesis of noroviruses. The small P particles may also be a useful alternative in study of norovirus-host interaction and vaccine development for noroviruses.

Norovirus P particle, a novel platform for vaccine development and antibody production.

The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli, extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.

Possible role of DMP1 in dentin mineralization.

Dentin Matrix Protein 1 (DMP1), the essential noncollagenous proteins in dentin and bone, is believed to play an important role in the mineralization of these tissues, although the mechanisms of its action are not fully understood. To gain insight into DMP1 functions in dentin mineralization we have performed immunomapping of DMP1 in fully mineralized rat incisors and in vitro calcium phosphate mineralization experiments in the presence of DMP1. DMP1 immunofluorescene was localized in peritubular dentin (PTD) and along the dentin-enamel boundary. In vitro phosphorylated DMP1 induced the formation of parallel arrays of crystallites with their c-axes co-aligned. Such crystalline arrangement is a hallmark of mineralized collagen fibrils of bone and dentin. Interestingly, in DMP1-rich PTD, which lacks collagen fibrils, the crystals are organized in a similar manner. Based on our findings we hypothesize, that in vivo DMP1 controls the mineral organization outside of the collagen fibrils and plays a major role in the mineralization of PTD.

Relationships between dentin and enamel mineral at the dentino-enamel boundary: electron tomography and high-resolution transmission electron microscopy study.

To better understand the nature of the relationships between mineral phases at the dentino-enamel boundary (DEB), we performed electron tomography (ET) and high-resolution transmission electron microscopy (HR-TEM) of the apical portions of rat incisors. The ET studies of the DEB at the secretory stage of amelogenesis revealed that nascent enamel crystals are co-aligned and closely associated with dentin crystallites in the mineralized von Korff fibers, with the distances between dentin and enamel crystals in the nanometer range. We have further studied the relationships between dentin and enamel crystals using HR-TEM lattice imaging of the DEB. Among dozens of high-resolution micrographs taken from the DEB we were able to identify only one case of lattice continuity between dentin and enamel crystals, indicating direct epitaxy. In other cases, although there was no direct continuity between the crystalline lattices, power spectra analysis of lattice images revealed a very high level of co-alignment between dentin and enamel crystals. Hence, we propose here that the high degree of alignment and integration between dentin and enamel mineral can be established either by epitaxy or without direct interactions between crystalline lattices, probably via regulation of mineral formation and organization by integrated organic matrices of dentin and enamel at the DEB.

Amelogenin-collagen interactions regulate calcium phosphate mineralization in vitro.

Collagen and amelogenin are two major extracellular organic matrix proteins of dentin and enamel, the mineralized tissues comprising a tooth crown. They both are present at the dentin-enamel boundary (DEB), a remarkably robust interface holding dentin and enamel together. It is believed that interactions of dentin and enamel protein assemblies regulate growth and structural organization of mineral crystals at the DEB, leading to a continuum at the molecular level between dentin and enamel organic and mineral phases. To gain insight into the mechanisms of the DEB formation and structural basis of its mechanical resiliency we have studied the interactions between collagen fibrils, amelogenin assemblies, and forming mineral in vitro, using electron microscopy. Our data indicate that collagen fibrils guide assembly of amelogenin into elongated chain or filament-like structures oriented along the long axes of the fibrils. We also show that the interactions between collagen fibrils and amelogenin-calcium phosphate mineral complexes lead to oriented deposition of elongated amorphous mineral particles along the fibril axes, triggering mineralization of the bulk of collagen fibril. The resulting structure was similar to the mineralized collagen fibrils found at the DEB, with arrays of smaller well organized crystals inside the collagen fibrils and bundles of larger crystals on the outside of the fibrils. These data suggest that interactions between collagen and amelogenin might play an important role in the formation of the DEB providing structural continuity between dentin and enamel.

Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than approximately 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing approximately 10(-4) of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (approximately 15 A), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.

Conformational flexibility facilitates self-assembly of complex DNA nanostructures.

Molecular self-assembly is a promising approach to the preparation of nanostructures. DNA, in particular, shows great potential to be a superb molecular system. Synthetic DNA molecules have been programmed to assemble into a wide range of nanostructures. It is generally believed that rigidities of DNA nanomotifs (tiles) are essential for programmable self-assembly of well defined nanostructures. Recently, we have shown that adequate conformational flexibility could be exploited for assembling 3D objects, including tetrahedra, dodecahedra, and buckyballs, out of DNA three-point star motifs. In the current study, we have integrated tensegrity principle into this concept to assemble well defined, complex nanostructures in both 2D and 3D. A symmetric five-point-star motif (tile) has been designed to assemble into icosahedra or large nanocages depending on the concentration and flexibility of the DNA tiles. In both cases, the DNA tiles exhibit significant flexibilities and undergo substantial conformational changes, either symmetrically bending out of the plane or asymmetrically bending in the plane. In contrast to the complicated natures of the assembled structures, the approach presented here is simple and only requires three different component DNA strands. These results demonstrate that conformational flexibility could be explored to generate complex DNA nanostructures. The basic concept might be further extended to other biomacromolecular systems, such as RNA and proteins.