Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we ...report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the "3-bases-in, 3-bases-out" conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123-139, which may serve as a valuable epitope for surveillance and diagnostics.
The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores ...(membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by “corking” the G20c portal protein into a SiN x nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid nanopore. Our integrated protein/solid-state device can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.
The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients ...following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4
and/or CD8
epitopes, including six immunodominant regions. Six optimized CD8
epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8
T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either ...membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.
Abstract
Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of ...human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.
The portal protein is a key component of many double-stranded DNA viruses, governing capsid assembly and genome packaging. Twelve subunits of the portal protein define a tunnel, through which DNA is ...translocated into the capsid. It is unknown how the portal protein functions as a gatekeeper, preventing DNA slippage, whilst allowing its passage into the capsid, and how these processes are controlled. A cryo-EM structure of the portal protein of thermostable virus P23-45, determined in situ in its procapsid-bound state, indicates a mechanism that naturally safeguards the virus against genome loss. This occurs via an inversion of the conformation of the loops that define the constriction in the central tunnel, accompanied by a hydrophilic-hydrophobic switch. The structure also shows how translocation of DNA into the capsid could be modulated by a changing mode of protein-protein interactions between portal and capsid, across a symmetry-mismatched interface.
Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and ...accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.
By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered ...N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.
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•X-ray structure of the 14-3-3/pHSPB6 heterotetrameric complex was obtained•N-terminal domain of HSPB6 becomes partially ordered within the complex•Synergistic binding explains effective sequestering of 14-3-3 by pHSPB6•First ever atomic resolution snapshot of a human small HSP in a functional state
Sluchanko et al. describe the first atomic structure of a full-length human small heat shock protein, HSPB6, determined within a heterotetrameric complex with the 14-3-3 protein, the universal signaling hub. Phosphorylation-dependent formation of this complex, a process involved in smooth muscle relaxation, results in stabilization of the highly intrinsically disordered N-terminal domain of HSPB6.
Abstract
Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed ...bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
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•SARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms.•This association with 14-3-3 strictly depends on phosphorylation of N.•The two proteins interact in 2:2 ...stoichiometry and with the KD in a μM range.•Affinity of interaction depends on the specific 14-3-3 isoform.•Conserved Ser197-phosphopeptide of N is critical for the interaction.
The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.
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