Significance Hepatitis B virus (HBV) is a major pathogen, yet no fully effective therapies exist. HBc is the multifunctional, capsid-forming protein essential for HBV replication. HBc structural plasticity is reportedly functionally important. We analyzed the folding mechanism of HBc using a multidisciplinary approach, including microscale thermophoresis and ion mobility spectrometry–mass spectrometry. HBc folds in a 3-state transition with a dimeric, helical intermediate. We found evidence of a strained native ensemble wherein the energy landscapes for folding, allostery, and capsid formation are linked. Mutations thermodynamically trapped HBc in conformations unable to form capsids, suggesting chemical chaperones could elicit similar, potentially antiviral, effects.

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

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The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21 1-552 (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21 1-552 trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21 1-552 trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21 1-552 are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (Ta CT-ACD and Ps CT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the Ta CT-ACD and Ps CT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Ta dimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHspsubstrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150-183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50-111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.

Background: We studied assembly of HBV capsids using a genetically-fused dimer.Results: Assembly intermediates composed of two, three and five copies of a fused dimer have been isolated.Conclusion: Capsid assembly could progress using dimeric, trimeric, and pentameric intermediates.Significance: The low abundance and transient nature of assembly intermediates make analysis challenging, however, the stability of the intermediates described here has overcome this.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ70 and σ54, the domain movements of the latter have evolved to require an activator ATPase.