Juliana R. Cortines

Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction

Proper assembly of viruses must occur through specific interactions between capsid proteins. Many double-stranded DNA viruses and bacteriophages require internal scaffolding proteins to assemble their coat proteins into icosahedral capsids. The 303 amino acid bacteriophage P22 scaffolding protein is mostly helical, and its C-terminal helix-turn-helix (HTH) domain binds to the coat protein during virion assembly, directing the formation of an intermediate structure called the procapsid. The interaction between coat and scaffolding protein HTH domain is electrostatic, but the amino acids that form the protein-protein interface have yet to be described. In the present study, we used alanine scanning mutagenesis of charged surface residues of the C-terminal HTH domain of scaffolding protein. We have determined that P22 scaffolding protein residues R293 and K296 are crucial for binding to coat protein and that the neighboring charges are not essential but do modulate the affinity between the two proteins.

α-Synuclein as an intrinsically disordered monomer – fact or artefact?

Fibrillization of the protein α‐synuclein (α‐syn) is a hallmark of Parkinsons disease and other α‐synucleinopathies. The well‐established idea that α‐syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that α‐syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for α‐syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant α‐syn in Escherichia coli. We demonstrate that α‐syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross‐linking protocol as previously suggested. Unlike the helically folded tetramer, α‐syn dimer is prone to fibrillate and thus it may be an interesting target for anti‐fibrillogenic molecules.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

ABSTRACT Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Unraveling the role of the C-terminal helix turn helix of the coat-binding domain of bacteriophage P22 scaffolding protein

Background: Viral scaffolding proteins interact with coat proteins to drive procapsid assembly. Results: Amino acid substitutions in the turn and between the helices of the coat protein-binding domain of scaffolding protein block procapsid assembly. Conclusion: The orientation of helices in the scaffolding helix turn helix domain is critical for procapsid assembly. Significance: Understanding scaffolding/coat protein interactions illuminates the mechanism of assembly of many large viruses. Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.

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

ABSTRACT 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 proteins 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 proteins 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.

The Importance of a Gatekeeper Residue on the Aggregation of Transthyretin IMPLICATIONS FOR TRANSTHYRETIN-RELATED AMYLOIDOSES

Background: Proteins have adopted negative design to diminish aggregation. Results: The replacement of Lys-35 by Leu increases the amyloidogenicity of the 26–57 segment of TTR as well as the entire protein. Conclusion: Lys-35 is as a gatekeeper residue in TTR, and its protective effect is suppressed by heparin. Significance: The elucidation of the principles that govern protein aggregation is helpful for the design of strategies against amyloid diseases. Protein aggregation into β-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26–57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26–57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.

Conformational dissection of Thermomyces lanuginosus lipase in solution

Lipases are triacyl glycerol acyl hydrolases, which catalyze hydrolysis of esters, esterification and transesterification reactions, among others. Some of these enzymes have a large hydrophobic pocket covered by an alpha-helical mobile surface loop (the lid). Protein-protein interactions can occur through adsorption of two open lids of individual lipases. We investigated the conformation and oligomeric state of Thermomyces lanuginosus lipase (TLL) in solution by spectroscopic and mass spectrometry techniques. Information about oligomerization of this important industrial enzyme is only available for TLL crystals; therefore, we have done a throughout investigation of the conformation of this lipase in solution. SDS-PAGE and mass spectrometry analysis of size-exclusion chromatography eluted fractions indicated the presence of both monomeric and dimeric populations of TLL. The stability of the enzyme upon thermal and guanidine hydrochloride treatment was examined by circular dichroism and fluorescence emission spectroscopy. Small angle x-ray scattering and ion mobility mass spectrometry analysis revealed that TLL is found as a mixture of monomers and dimers at the assayed concentrations. Although previous x-ray diffraction data showed TLL as a dimer in the crystal (PDB: 1DT3), to our knowledge our report is the first evidencing that TLL co-exists as stable dimeric and monomeric forms in solution.

A viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly

A novel role for scaffolding protein in portal ring formation. Most double-stranded DNA viruses package genetic material into empty precursor capsids (or procapsids) through a dodecameric portal protein complex that occupies 1 of the 12 vertices of the icosahedral lattice. Inhibiting incorporation of the portal complex prevents the formation of infectious virions, making this step an excellent target for antiviral drugs. The mechanism by which a sole portal assembly is selectively incorporated at the special vertex is unclear. We recently showed that, as part of the DNA packaging process for bacteriophage P22, the dodecameric procapsid portal changes conformation to a mature virion state. We report that preformed dodecameric rings of P22 portal protein, as opposed to portal monomers, incorporate into nascent procapsids, with preference for the procapsid portal conformation. Finally, a novel role for P22 scaffolding protein in triggering portal ring formation from portal monomers is elucidated and validated by incorporating de novo assembled portal rings into procapsids.

UV-induced selective oxidation of Met5 to Met-sulfoxide leads to the formation of neurotoxic fibril-incompetent α-synuclein oligomers

Abstract Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinsons disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the 1H-15N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.

Microscopic Characterization of the Brazilian Giant Samba Virus

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses.