Advanced sampling simulations of coupled folding and binding of phage P22 N-peptide to boxB RNA.

Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules' spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA's center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.

Bioinspired Approaches to Self-Assembly of Virus-like Particles: From Molecules to Materials.

When viewed through the lens of materials science, nature provides a vast library of hierarchically organized structures that serve as inspiration and raw materials for new synthetic materials. The structural organization of complex bioarchitectures with advanced functions arises from the association of building blocks and is strongly supported by ubiquitous mechanisms of self-assembly, where interactions among components result in spontaneous assembly into defined structures. Viruses are exemplary, where a capsid structure, often formed from the self-assembly of many individual protein subunits, serves as a vehicle for the transport and protection of the viral genome. Higher-order assemblies of viral particles are also found in nature with unexpected collective behaviors. When the infectious aspect of viruses is removed, the self-assembly of viral particles and their potential for hierarchical assembly become an inspiration for the design and construction of a new class of functional materials at a range of different length scales.Salmonella typhimurium bacteriophage P22 is a well-studied model for understanding viral self-assembly and the construction of virus-like particle (VLP)-based materials. The formation of cage-like P22 VLP structures results from scaffold protein (SP)-directed self-assembly of coat protein (CP) subunits into icosahedral capsids with encapsulation of SP inside the capsid. Employing the CP-SP interaction during self-assembly, the encapsulation of guest protein cargos inside P22 VLPs can be achieved with control over the composition and the number of guest cargos. The morphology of cargo-loaded VLPs can be altered, along with changes in both the physical properties of the capsid and the cargo-capsid interactions, by mimicking aspects of the infectious P22 viral maturation. The structure of the capsid differentiates the inside cavity from the outside environment and serves as a protecting layer for the encapsulated cargos. Pores in the capsid shell regulate molecular exchange between inside and outside, where small molecules can traverse the capsid freely while the diffusion of larger molecules is limited by the pores. The interior cavity of the P22 capsid can be packed with hundreds of copies of cargo proteins (especially enzymes), enforcing intermolecular proximity, making this an ideal model system in which to study enzymatic catalysis in crowded and confined environments. These aspects highlight the development of functional nanomaterials from individual P22 VLPs, through biomimetic design and self-assembly, resulting in fabrication of nanoreactors with controlled catalytic behaviors.Individual P22 VLPs have been used as building blocks for the self-assembly of higher-order structures. This relies on a balance between the intrinsic interparticle repulsion and a tunable interparticle attraction. The ordering of VLPs within three-dimensional assemblies is dependent on the balance between repulsive and attractive interactions: too strong an attraction results in kinetically trapped disordered structures, while decreasing the attraction can lead to more ordered arrays. These higher-order assemblies display collective behavior of high charge density beyond those of the individual VLPs.The development of synthetic nanomaterials based on P22 VLPs demonstrates how the potential for hierarchical self-assembly can be applied to other self-assembling capsid structures across multiple length scales toward future bioinspired functional materials.

Higher-Order VLP-Based Protein Macromolecular Framework Structures Assembled via Coiled-Coil Interactions.

Hierarchical organization is one of the fundamental features observed in biological systems that allows for efficient and effective functioning. Virus-like particles (VLPs) are elegant examples of a hierarchically organized supramolecular structure, where many subunits are self-assembled to generate the functional cage-like architecture. Utilizing VLPs as building blocks to construct two- and three-dimensional (3D) higher-order structures is an emerging research area in developing functional biomimetic materials. VLPs derived from P22 bacteriophages can be repurposed as nanoreactors by encapsulating enzymes and modular units to build higher-order catalytic materials via several techniques. In this study, we have used coiled-coil peptide interactions to mediate the P22 interparticle assembly into a highly stable, amorphous protein macromolecular framework (PMF) material, where the assembly does not depend on the VLP morphology, a limitation observed in previously reported P22 PMF assemblies. Many encapsulated enzymes lose their optimum functionalities under the harsh conditions that are required for the P22 VLP morphology transitions. Therefore, the coiled-coil-based PMF provides a fitting and versatile platform for constructing functional higher-order catalytic materials compatible with sensitive enzymes. We have characterized the material properties of the PMF and utilized the disordered PMF to construct a biocatalytic 3D material performing single- and multistep catalysis.

Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22.

Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

Conservation and Divergence of the I-Domain Inserted into the Ubiquitous HK97 Coat Protein Fold in P22-Like Bacteriophages.

Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel ß-barrels. Nevertheless, significant structural differences occur in loops connecting the ß-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions.IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded ß-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages.

A Hydrophobic Network: Intersubunit and Intercapsomer Interactions Stabilizing the Bacteriophage P22 Capsid.

Double-stranded DNA (dsDNA) tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here, we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra- and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra- and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Enclosed in a 23 MDa Procapsid.

Scaffolding proteins (SPs) are required for the capsid shell assembly of many tailed double-stranded DNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, only one high-resolution structure is available for SPs within procapsids. Here, we use the inherent size limit of NMR to identify mobile segments of the 303-residue phage P22 SP free in solution and when incorporated into a ∼23 MDa procapsid complex. Free SP gives NMR signals from its acidic N-terminus (residues 1-40) and basic C-terminus (residues 264-303), whereas NMR signals from the middle segment (residues 41-263) are missing because of intermediate conformational exchange on the NMR chemical shift timescale. When SP is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix domain disappear because of binding to the procapsid interior. Signals from the N-terminal domain persist, indicating that this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus, coupled with its high content of negative charges, is likely important for dissociation and release of SP during the double-stranded DNA genome packaging step accompanying phage maturation.

Development of target-tunable P22 VLP-based delivery nanoplatforms using bacterial superglue.

Protein cage nanoparticles are widely used as targeted delivery nanoplatforms, because they have well-defined symmetric architectures, high biocompatibility, and enough plasticity to be modified to produce a range of different functionalities. Targeting peptides and ligands are often incorporated on the surface of protein cage nanoparticles. In this research, we adopted the SpyTag/SpyCatcher protein ligation system to covalently display target-specific affibody molecules on the exterior surface of bacteriophage P22 virus-like particles (VLP) and evaluated their modularity and efficacy of targeted delivery. We genetically introduced the 13 amino acid SpyTag peptide into the C-terminus of the P22 capsid protein to construct a target-tunable nanoplatform. We constructed two different SpyCatcher-fused affibody molecules as targeting ligands, SC-EGFRAfb and SC-HER2Afb, which selectively bind to EGFR and HER2 surface markers, respectively. We produced target-specific P22 VLP-based delivery nanoplatforms for the target cell lines by selectively combining SpyTagged P22 VLP and SC-fused affibody molecules. We confirmed its target-switchable modularity through cell imaging and verified the target-specific drug delivery efficacy of the affibody molecules displaying P22 VLP using cell viability assays. The P22 VLP-based delivery nanoplatforms can be used as multifunctional delivery vehicles by ligating other functional proteins, as well as affibody molecules. The interior cavity of P22 VLP can be also used to load cargoes like enzymes and therapeutic proteins. We anticipate that the nanoplatforms will provide new opportunities for developing target-specific functional protein delivery systems.

Chemically Induced Morphogenesis of P22 Virus-like Particles by the Surfactant Sodium Dodecyl Sulfate.

In the infectious P22 bacteriophage, the packaging of DNA into the initially formed procapsid triggers a remarkable morphological transformation where the capsid expands from 58 to 62 nm. Along with the increase in size, this maturation also provides greater stability to the capsid and initiates the release of the scaffolding protein (SP). (2,4) In the P22 virus-like particle (VLP), this transformation can be mimicked in vitro by heating the procapsid particles to 65 °C or by treatment with sodium dodecyl sulfate (SDS). (5,6) Heating the P22 particles at 65 °C for 20 min is well established to trigger the transformation of P22 to the expanded (EX) P22 VLP but does not always result in a fully expanded population. Incubation with SDS resulted in a >80% expanded population for all P22 variants used in this work. This study elucidates the importance of the stoichiometric ratio between P22 subunits and SDS, the charge of the headgroup, and length of the carbon chain for the transformation. We propose a mechanism by which the expansion takes place, where both the negatively charged sulfate group and hydrophobic tail interact with the coat protein (CP) monomers within the capsid shell in a process that is facilitated by an internal osmotic pressure generated by an encapsulated macromolecular cargo.

Function of bacteriophage G7C esterase tailspike in host cell adsorption.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation.

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.

A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain.

P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage.

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly.

UNLABELLED: Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. IMPORTANCE: Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.

Measurement of the accurate mass of a 50 MDa infectious virus.

RATIONALE: Bacteriophage P22 is believed to contain a total of 521 copies of 9 different proteins and a 41,724 base pair genome. Despite its enormous size and complexity, phage P22 can be electrosprayed, and it remains intact in ultra-high vacuum where its molar mass distribution has been measured. METHODS: Phage P22 virions were generated by complementation in Salmonella enterica and purified. They were transferred into 100 mM ammonium acetate and then electrosprayed. The masses of individual virions were determined using charge detection mass spectrometry. RESULTS: The stoichiometry of the protein components of phage P22 is sufficiently well known that the theoretical molar mass can be determined to within a narrow range. The measured average molar mass of phage P22, 52,180 ± 59 kDa, is consistent with the theoretical molar mass and supports the proposed stoichiometry of the components. The intrinsic width of the phage P22 mass distribution can be accounted for by the distribution of DNA packaged by the headful mechanism. CONCLUSIONS: At over 50 MDa, phage P22 is the largest object with a well-defined molar mass to be analyzed by mass spectrometry. The narrow measured mass distribution indicates that the virions survive the transition into the gas phase intact. Copyright © 2016 John Wiley & Sons, Ltd.

Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

UNLABELLED: Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. IMPORTANCE: Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery.

BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker.

Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection.

Exploring the atomic structure and conformational flexibility of a 320†Šlong engineered viral fiber using X-ray crystallography.

Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Šresolution crystal structure of this engineered fiber, which measures ∼320 Šin length and is only 20-35 Šwide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility.