Cameron Haase-Pettingell

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates.

An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off‐pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel β coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Tem‐ perature‐sensitive folding (tsf) mutations identify sites in the β coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins.—King, J., Haase‐Pettingell, C., Robinson, A. S., Speed, M., Mitraki, A. Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates. FASEB J. 10, 57‐66 (1996)

Visualizing virus assembly intermediates inside marine cyanobacteria

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Visualizing the structural changes of bacteriophage epsilon15 and its Salmonella host during infection

The efficient mechanism by which double-stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single-particle electron cryomicroscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (ɛ15) and P-SSP7. We have used electron cryotomography and three-dimensional subvolume averaging to capture snapshots of ɛ15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of ɛ15 attach to the surface of the host cell. Next, ɛ15s tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.

Mutational effects on inclusion body formation.

Publisher Summary Inclusion body formation has been studied most extensively in Escherichia coli ( E. coli) and Salmonella typhimurium , but the phenomenon is not isolated to prokaryotes. Inclusion bodies have been detected in eukaryotic cells, for example, in diseased human hepatocytes and inside algal chloroplasts. In bacteria, inclusion bodies have been observed to span the full width of a cell. The recognition of the nature of aggregation upon dilution from denaturant left open the question of the origin of inclusion bodies formed from newly synthesized proteins within cells. Intracellular protein deposits, such as Heinz bodies had been identified as associated with specific amino acid substitutions, but these findings from pathology were not initially recognized as protein misfolding. Investigation of the intracellular folding of the P22 tail spike provided direct evidence, that inclusion bodies were derived from the in vivo association of partially folded intermediates. Studies with interferon and interleukin expressed in E. coli confirmed that single amino acid substitutions could influence misfolding and aggregation pathways. This chapter reviews these efforts to isolate mutations affecting intracellular chain folding and other studies related to them.

Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling

High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3–5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit–subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages.

The interdigitated β-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right‐handed parallel β‐helices (residues 143–540), pack side‐by‐side around the three‐fold axis. After residue 540, these single chain β‐helices terminate and residues 541–567 of the three polypeptide chains wrap around each other to form a three‐stranded interdigitated β‐helix. Three mutants located in this region — G546D, R563Q, and A575T — blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially‐folded protrimer to the native trimer. The protrimer‐like species accumulating in the G546D mutant melted out at 42°C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in β‐helix folding. The simplest interpretation of these results is that the very high thermostability (Tm = 88°C), protease and detergent resistance of the native tailspike acquired in the protrimer‐to‐trimer transition, depends on the formation of the three‐stranded interdigitated region. This interdigitated β‐helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Background: The subunit-specific roles of the CCT subunits in the chaperonin, TRiC, have not been elucidated. Results: When expressed in E. coli, CCT4 and CCT5 form TRiC-like homo-oligomeric rings. Conclusion: TRiC does not require all eight CCT subunits to form functional rings. Significance: The unexpected formation of homo-oligomeric CCT rings provides clues into the assembly of TRiC as a complex. Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

Accurate model annotation of a near-atomic resolution cryo-EM map

Significance Electron cryomicroscopy is a rapidly growing field for macromolecular structure determination. We establish a computational protocol to construct a de novo atomic model from a cryo-EM density map, along with associated metadata that describe coordinate uncertainty and the density at each atom. This model faithfully replicates experimental map densities, as evidenced by cross-correlation and other metrics. Our method of annotation will be especially informative for macromolecular assemblies that exhibit resolvability variations in different parts of their structure. This procedure was applied to a 3.3-Å-resolution structure of the P22 bacteriophage to delineate interactions that stabilize the neighboring subunits in a T = 7 icosahedral capsid. Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.

Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike

The predominantly β‐sheet phage P22 tailspike adhesin contains eight reduced cysteines per 666 residue chain, which are buried and unreactive in the native trimer. In the pathway to the native trimer, both in vivo and in vitro transient interchain disulfide bonds are formed and reduced. This occurs in the protrimer, an intermediate in the formation of the interdigitated β‐sheets of the trimeric tailspike. Each of the eight cysteines was replaced with serine by site‐specific mutagenesis of the cloned P22 tailspike gene and the mutant genes expressed in Escherichia coli. Although the yields of native‐like Cys>Ser proteins varied, sufficient soluble trimeric forms of each of the eight mutants accumulated to permit purification. All eight single Cys>Ser mature proteins maintained the high thermostability of the wild type, as well as the wild‐type biological activity in forming infectious virions. Thus, these cysteine thiols are not required for the stability or activity of the native state. When their in vivo folding and assembly kinetics were examined, six of the mutant substitutions—C267S, C287S, C458S, C613S, and C635S—were significantly impaired at higher temperatures. Four—C290S, C496, C613S, and C635—showed significantly impaired kinetics even at lower temperatures. The in vivo folding of the C613S/C635S double mutant was severely defective independent of temperature. Since the trimeric states of the single Cys>Ser substituted chains were as stable and active as wild type, the impairment of tailspike maturation presumably reflects problems in the in vivo folding or assembly pathways. The formation or reduction of the transient interchain disulfide bonds in the protrimer may be the locus of these kinetic functions.