Gregory J. Bedwell

Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

Charge detection mass spectrometry of bacteriophage P22 procapsid distributions above 20 MDa

RATIONALE Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids. METHODS P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution. RESULTS The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges. CONCLUSIONS The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa.

Structural and Functional Characterization of the Mumps Virus Phosphoprotein

ABSTRACT The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsid-like particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N- and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (POD) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV POD is formed by one pair of long parallel α-helices with another pair in opposite orientation. Unlike the parallel orientation of each α-helix in the tetramer of Sendai virus POD, this represents a novel orientation of a POD where both the N- and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N- and the C-terminal domains are involved in binding the nucleocapsid.

Identification of eRF1 residues that play critical and complementary roles in stop codon recognition

The initiation and elongation stages of translation are directed by codon-anticodon interactions. In contrast, a release factor protein mediates stop codon recognition prior to polypeptide chain release. Previous studies have identified specific regions of eukaryotic release factor one (eRF1) that are important for decoding each stop codon. The cavity model for eukaryotic stop codon recognition suggests that three binding pockets/cavities located on the surface of eRF1s domain one are key elements in stop codon recognition. Thus, the model predicts that amino acid changes in or near these cavities should influence termination in a stop codon-dependent manner. Previous studies have suggested that the TASNIKS and YCF motifs within eRF1 domain one play important roles in stop codon recognition. These motifs are highly conserved in standard code organisms that use UAA, UAG, and UGA as stop codons, but are more divergent in variant code organisms that have reassigned a subset of stop codons to sense codons. In the current study, we separately introduced TASNIKS and YCF motifs from six variant code organisms into eRF1 of Saccharomyces cerevisiae to determine their effect on stop codon recognition in vivo. We also examined the consequences of additional changes at residues located between the TASNIKS and YCF motifs. Overall, our results indicate that changes near cavities two and three frequently mediated significant effects on stop codon selectivity. In particular, changes in the YCF motif, rather than the TASNIKS motif, correlated most consistently with variant code stop codon selectivity.

Higher-Order Structure of the Rous Sarcoma Virus SP Assembly Domain

ABSTRACT Purified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly. IMPORTANCE The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

Efficient transcription by RNA polymerase I using recombinant core factor.

Transcription of ribosomal DNA by RNA polymerase I is a central feature of eukaryotic ribosome biogenesis. Since ribosome synthesis is closely linked to cell proliferation, there is a need to define the molecular mechanisms that control transcription by RNA polymerase I. To fully define the factors that control RNA polymerase I activity, biochemical analyses using purified transcription factors are essential. Although such assays exist, one limitation is the low abundance and difficult purification strategies required for some of the essential transcription factors for RNA polymerase I. Here, we describe a new method for expression and purification of the three subunit core factor complex from Escherichia coli. We demonstrate that the recombinant material is more active than yeast-derived core factor in assays for RNA polymerase I transcription in vitro. Finally, we use recombinant core factor to differentiate between two opposing models for the role of the TATA-binding protein in transcription by RNA polymerase I.

Formation mechanism of chalcogenide nanocrystals confined inside genetically engineered virus-like particles.

Engineered virus-like particles (VLP) are attractive for fabricating nanostructured materials for applications in diverse areas such as catalysis, drug delivery, biomedicine, composites, etc. Basic understanding of the interaction between the inorganic guest and biomolecular host is thus important for the controlled synthesis of inorganic nanoparticles inside VLP and rational assembly of ordered VLP-based hierarchical nanostructures. We have investigated in detail the formation mechanism and growth kinetics of semiconducting nanocrystals confined inside genetically engineered bacteriophage P22 VLP using semiconducting CdS as a prototypical example. The selective nucleation and growth of CdS at the engineered sites is found to be uniform during the early stage, followed by a more stochastic growth process. Furthermore, kinetic studies reveal that the presence of an engineered biotemplate helps in significantly retarding the reaction rate. These findings provide guidance for the controlled synthesis of a wide range of other inorganic materials confined inside VLP, and are of practical importance for the rational design of VLP-based hierarchical nanostuctures.

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

Background: Calmodulin (CaM) is recruited into the death-inducing signaling complex in cholangiocarcinoma cells. Results: CaM binds to FasDD in a 2:1 CaM:FasDD model. CaM antagonists abolish FasDD-CaM interactions. Conclusion: Data offer a structural basis for Fas-CaM interactions and mechanisms of inhibition. Significance: Elucidating the structural determinants of Fas-CaM interaction is critical to understanding the functional role of CaM in Fas-mediated apoptosis. The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ∼2 μm and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Selective Biotemplated Synthesis of TiO2 Inside a Protein Cage

Biological organisms have evolved tremendous control over the synthesis of inorganic materials in aqueous solutions at standard conditions. Such control over material properties is difficult to achieve with current synthesis strategies. Biotemplated synthesis of materials has been demonstrated to be efficient at facilitating the formation of various inorganic species. In this study, we employ a protein cage-based system to synthesize photoactive TiO2 nanoparticles less than 10 nm in diameter. We also demonstrate phase control over the material, with the ability to synthesize both anatase and rutile TiO2 using distinct biomineralization peptides within the protein cage. Finally, using analytical ultracentrifugation, we are able to resolve distinct reaction products and approximate their loading. We find that two distinct species comprise the reaction products, likely representing procapsid-like particles with early, precursor metal oxide clusters, and shells nearly full with crystalline TiO2 nanoparticles, respectively.