Browsing by Author "Tao, Yizhi Jane"

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    Assessing Photocatalytic Oxidation Using Modified TiO2 Nanomaterials for Virus Inactivation in Drinking Water: Mechanisms and Application
    (2013-06-05) Liga, Michael; Li, Qilin; Alvarez, Pedro J.; Barron, Andrew R.; Tao, Yizhi Jane
    Photocatalytic oxidation is an alternative water treatment method under consideration for disinfecting water. Chlorine disinfection can form harmful byproducts, and some viruses (e.g. adenoviruses) are resistant to other alternative disinfection methods. Photocatalytic oxidation using nano-sized photocatalytic particles (e.g. TiO2, fullerene) holds promise; however, it is limited by its low efficiency and long required treatment times. This research focuses on improving virus inactivation by photocatalytic oxidation by modifying catalysts for improved activity, by analyzing virus inactivation kinetics, and by elucidating the inactivation mechanisms of adenovirus serotype 2 (AdV2) and bacteriophage MS2. Modifying TiO2 with silver (nAg/TiO2) or silica (SiO2-TiO2) improves the inactivation kinetics of bacteriophage MS2 by a factor of 3-10. nAg/ TiO2 increases hydroxyl radical (HO•) production while SiO2 increases the adsorption of MS2 to TiO2. These results suggest that modifying the photocatalyst surface to increase contaminant adsorption is an important improvement strategy along with increasing HO• production. The inactivation kinetics of AdV2 by P25 TiO2 is much slower than the MS2 inactivation kinetics and displays a strong shoulder, which is not present in the MS2 kinetics. nAg/TiO2 initially improves the inactivation rate of AdV2. SiO2-TiO2 reduces the AdV2 inactivation kinetics since adsorption is not significantly enhanced, as it is with MS2. Amino-C60 is highly effective for AdV2 inactivation under visible light irradiation, making it a good material for use in solar disinfection systems. The efficacy of amino-fullerene also demonstrates that singlet oxygen is effective for AdV2 inactivation. When exposed to irradiated TiO2, AdV2 hexon proteins are heavily damaged resulting in the release of DNA. DNA damage is also present but may occur after capsids break. With MS2, the host interaction protein is rapidly damaged, but not the coat protein. The kinetics of MS2 inactivation are rapid since it may quickly lose its ability to attach to host cells, while AdV2 kinetics are slower since the entire capsid must undergo heavy oxidation before inactivation occurs. Adenovirus inactivation likely occurs through breaching the capsid followed by radical attack of DNA and core proteins.
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    Biochemical and Structural Evidence in Support of a Coherent Model for the Formation of the Double-Helical Influenza A Virus Ribonucleoprotein
    (American Society for Microbiology, 2012) Ye, Qiaozhen; Guu, Tom S.Y.; Mata, Dougslas A.; Kuo, Rei-Lin; Smith, Bartram; Krug, Robert M.; Tao, Yizhi Jane
    Influenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays.
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    Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2
    (Public Library of Science, 2013) Zhang, Nenggang; Jiang, Yunyun; Mao, Qilong; Demeler, Borries; Tao, Yizhi Jane; Pati, Debananda
    The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ﾒcoreﾒ cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301ﾖ750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located At 60-81 aa of the N-terminus and 383ﾖ392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, a-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this ahelical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SAbinding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.
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    Crystal Structure of the Human Astrovirus Capsid Protein
    (American Association for Microbiology, 2016) Toh, Yukimatsu; Harper, Justin; Dryden, Kelly A.; Yeager, Mark; Arias, Carlos F.; Méndez, Ernesto; Tao, Yizhi Jane
    Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.
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    Data publication with the structural biology data grid supports live analysis
    (Springer Nature, 2016) Meyer, Peter A.; Socias, Stephanie; Key, Jason; Ransey, Elizabeth; Tjon, Emily C.; Buschiazzo, Alejandro; Lei, Ming; Botka, Chris; Withrow, James; Neau, David; Rajashankar, Kanagalaghatta; Anderson, Karen S.; Baxter, Richard H.; Blacklow, Stephen C.; Boggon, Titus J.; Bonvin, Alexandre M.J.J.; Borek, Dominika; Brett, Tom J.; Caflisch, Amedeo; Chang, Chung-I; Chazin, Walter J.; Corbett, Kevin D.; Cosgrove, Michael S.; Crosson, Sean; Dhe-Paganon, Sirano; Di Cera, Enrico; Drennan, Catherine L.; Eck, Michael J.; Eichman, Brandt F.; Fan, Qing R.; Ferré-D'Amaré, Adrian R.; Fromme, J.Christopher; Garcia, K.Christopher; Gaudet, Rachelle; Gong, Peng; Harrison, Stephen C.; Heldwein, Ekaterina E.; Jia, Zongchao; Keenan, Robert J.; Kruse, Andrew C.; Kvansakul, Marc; McLellan, Jason S.; Modis, Yorgo; Nam, Yunsun; Otwinowski, Zbyszek; Pai, Emil F.; Pereira, Pedro José Barbosa; Petosa, Carlo; Raman, C.S.; Rapoport, Tom A.; Roll-Mecak, Antonina; Rosen, Michael K.; Rudenko, Gabby; Schlessinger, Joseph; Schwartz, Thomas U.; Shamoo, Yousif; Sondermann, Holger; Tao, Yizhi Jane; Tolia, Niraj H.; Tsodikov, Oleg V.; Westover, Kenneth D.; Wu, Hao; Foster, Ian; Fraser, James S.; Maia, Filipe R.N.C.; Gonen, Tamir; Kirchhausen, Tom; Diederichs, Kay; Crosas, Mercè; Sliz, Piotr
    Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.
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    Design and Structural Characterization of Self-Assembling Triple Helical Heterotrimers
    (2013-06-05) Fallas Valverde, Jorge; Hartgerink, Jeffrey D.; Wolynes, Peter G.; Tao, Yizhi Jane
    Design of self-assembling ABC-type collagen triple helical heterotrimers is challenging due to the number of competing species that can be formed in ternary mixture of peptides with a high propensity to fold into triple helices and the fact that well understood rules for pair-wise amino acid stabilization of the canonical collagen triple helix have remained elusive. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population towards a desired target but the presence of competing states in mixtures has remained an outstanding problem. In this work we use high-resolution structural biology techniques to do a detailed study of stabilizing pair-wise interactions between positively and negatively charged amino acids in triple helices. Two types of contacts with distinct sequence requirements depending on the relative stagger of the interacting chains are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. Using this knowledge we developed distinct design strategies to improve the specificity of our designed systems towards the desired ABC heterotrimeric target state. We validate our strategies through the synthesis and characterization of the designed sequences and show that they self-assemble into a highly stable ABC triple helices with control over composition in the case of the rational approach and with control over both composition and register in the case of the computational approach.
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    Design of Heterotrimeric Collagen Triple Helices
    (2014-04-24) Jalan, Abhishek; Hartgerink, Jeffrey D.; Wolynes, Peter G.; Tao, Yizhi Jane
    Select loci in native collagen display clusters of contiguous amino acids that recognize a diverse array of extracellular matrix (ECM) and blood serum proteins critical for homeostasis and hemostasis. The mechanism of collagen binding to these proteins has primarily been elucidated using short peptides, called collagen mimetic peptides (CMPs), that independently fold into the so called homotrimeric collagen triple helices, where all three peptide chains have identical amino acid sequence. However, the homotrimer binding mechanism cannot be extrapolated to explain protein binding in AAB and ABC-type heterotrimeric collagens that contain either two or three unique polypeptide chains without significant speculation. Given the requirement of a one amino acid offset between the peptide chains in a collagen triple helix, a mixture of two or three unique peptides can self-assemble into 8 and 27 competing triple helices, respectively. Heterotrimeric CMPs have remained synthetically inaccessible due to the challenge associated with introducing bias in this ensemble of competing states. Previously, Hartgerink lab employed axial Lys – Asp / Glu salt-bridges to successfully self-assemble an ABC heterotrimer. Here, we extend this paradigm to successfully demonstrate the design of a proof-of-principle AAB heterotrimer. Four AAB heterotrimers, each carrying unpaired Lys, Asp or Glu and a combination of Lys – Asp or Lys – Glu axial salt-bridges, were designed. Of these, only the heterotrimer containing unpaired Glu and a combination of Lys – Asp as well as Lys – Glu salt-bridges successfully self-assembled into an AAB heterotrimer. Next, a general methodology to self-assemble AAB heterotrimers containing the α2β1 and α1β1-integrin recognition sequences from collagen I and IV, respectively, and the matrix metalloproteinase-1 cleavage sequence from collagen I was developed. The protein recognition sequences were included as guests in a host peptide sequence containing a network of salt-bridges that bias the ensemble of competing triple helices to the desired AAB heterotrimer. Successful self-assembly of heterotrimers across multiple guest sequences was observed, which demonstrates the wide applicability of the host sequence design. In future, binding of these heterotrimers to the ECM and blood serum proteins has the potential to unravel the mechanism of disease evolution across multiple disease settings. We also extended the salt-bridge based design paradigm to synthesize a new class of CMP constructs. Hydroxyproline-free collagen triple helices are lucrative for expression in bacterial systems. Using a combination of Lys – Asp and Lys – Glu salt-bridges, a hydroxyproline-free ABC collagen heterotrimer was successfully designed. Remarkably, this ABC heterotrimer was stable despite one the peptides containing no proline or hydroxyproline, a requirement previously thought to be critical for stability. Additionally, an ABC heterotrimer containing a non-canonical four residue offset between the peptide chains was designed. In this heterotrimer, the non-covalent interactions at the termini are unsatisfied which renders them “sticky” to further assembly. This design lays the groundwork to create longer and therefore, stickier offsets to facilitate self-assembly of collagen-mimetic nanofibers.
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    Evolutionary Trajectories to Daptomycin Resistance in Enterococcus faecalis
    (2013-11-18) Miller, Corwin; Olson, John S.; Shamoo, Yousif; Bennett, Matthew R.; Rudolf, Volker H. W.; Tao, Yizhi Jane
    With increasing amounts of hospital-acquired antibiotic resistant infections each year and staggering healthcare costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. In this thesis, I have combined experimental evolution, comparative whole genome sequencing, and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin resistant clinical pathogen Enterococcus faecalis strain S613. Maintaining cells inside a turbidostat, a single polymorphic culture was grown sustaining both planktonic and non-planktonic (e.g. biofilm) populations in co-culture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. This approach revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Genetic and phenotypic comparisons between resistant isolates also identified convergent evolutionary trajectories, suggesting a common biochemical mechanism of resistance. Despite the relative ecological simplicity of this approach compared to the complexity of the human body, I show that experimental evolution can be used to rapidly identify clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.
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    Expression and Assembly of Human Picobirnavirus (hPBV) Using a Plasmid-Based Expression System
    (2023-04-05) Ouyang, Yu; Tao, Yizhi Jane; Gustin, Michael C.
    Picobirnavirus (PBV) is a small (35 nm in diameter, i.e., “pico”), non-enveloped, bi-segmented dsRNA (i.e., “bi-RNA”) virus. Since the discovery of PBV in 1988, PBV has been detected in various host species, including humans, mammals, birds, reptiles, and even in environments like sewage. The actual host of PBV remains controversial, and no infection model has been established to unveil the pathology or pathogenesis associated with PBV infection. In this study, I constructed a plasmid-based expression system for hPBV by expressing the two viral RNA segments in the E. coli Rosetta 2 strain. All predicted viral proteins, i.e., ORF1, ORF2, CP, and RdRP, were detected upon the viral RNA expression, indicating efficient translation from inherent ribosomal binding sites. Purified recombinant hPBV capsids were found to comprise: both viral RNA segments, ORF2, and RdRP. Viral RNA segments were preferentially packaged into the capsids over host mRNAs, presumably due to packaging signal (PS) sequences at the terminal ends. Such terminal PS sequences were able to carry a non-viral RNA sequence into the recombinant hPBV. ORF2 protein was observed in the VLPs for the first time. By engineering a series of truncation mutants, it was determined that the N- terminal domain of ORF2 is responsible for its incorporation into the capsids. Through immunoprecipitation assays, it was found that hPBV RdRP directly interacts with CP into assembly intermediates. Still, RdRP was only present at deficient levels in assembled capsids at its overexpression. This work presents a plasmid-based expression system to fully dissect molecular determinants for hPBV assembly and genome packaging. Results from this study indicate that hPBV is indeed a prokaryotic virus, but E. coli is likely not the natural host of hPBV. Co-immunoprecipitation of recombinant hPBV with lysates of different microbiota species in future studies would reveal the identity of the hPBV native host and establish the PBV infection model.
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    Force Activation of a Multimeric Adhesive Protein through Domain Conformational Change
    (2013-07-24) Wijeratne, Sitara; Kiang, Ching-Hwa; Tao, Yizhi Jane; Kono, Junichiro
    The force-induced activation of adhesive proteins such as von Willebrand Factor (VWF), which experience high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates pVWF multimers to bind platelets. Here we showed that a pathological level of high shear flow exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.
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    Functional and structural studies of influenza B virus hemagglutinin
    (2013-09-16) Ni, Fengyun; Ma, Jianpeng; Tao, Yizhi Jane; Matthews, Kathleen S.
    Influenza A and B viruses are major causes of seasonal flu epidemics each year. Hemagglutinin (HA) mediates the binding of virus to host cell and the fusion with host membrane. The crystal of HA in complex with antibody that reveals the mechanism by which antibody recognizes HA may not diffract to high resolution, thereby preventing the accurate interpretation of the structural model. The application of normal mode refinement that aims for improving the structure quality at the low resolution is tested. These studies provide some guidelines for future refinement of HA-antibody complex structures. By comparing the residues constituting the base of the receptor binding site of influenza A and B virus HAs, it is found that they share some similarities, except for a Phe at position 95 of influenza B virus hemagglutinin (BHA) versus Tyr in of influenza A virus hemagglutinin (AHA). The recombinant protein BHA containing the F95Y mutation exhibits the increased receptor binding affinity and specificity. However, recombinant viruses with the Phe95Tyr mutation show lower erythrocyte agglutination titer and decreased binding abilities with different cell lines. The replication of the Phe95Tyr mutant virus in mice is also attenuated. These data suggest that the increased receptor binding ability of HA alone is not advantageous to the pathogenesis of the viruses. The structure of BHA2 (a portion of BHA near the C-terminus) at the post-fusion state has been determined to 2.45 Å resolution. This protein forms a hairpin-like conformation rich in -helices. About 70 residues from the N-terminus is a three-stranded coiled coil, and the remaining of the protein packs in anti-parallel against the groove formed by the central helices. In the post-fusion state of BHA2, the helix converted from the B-loop in pre-fusion state contacts the C-terminal fragment of this protein with more hydrophobic interactions as compared to AHA2. This structure illustrates the distinct stabilization strategy employed by BHA2 to form a post-fusion state that resembles that for AHA2. These studies will further the understanding of BHA with respect to its role in receptor binding ability and fusion.
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    Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA–DNA pairing
    (Oxford University Press, 2016) Lin, Jiangguo; Countryman, Preston; Chen, Haijiang; Pan, Hai; Fan, Yanlin; Jiang, Yunyun; Kaur, Parminder; Miao, Wang; Gurgel, Gisele; You, Changjiang; Piehler, Jacob; Kad, Neil M.; Riehn, Robert; Opresko, Patricia L.; Smith, Susan; Tao, Yizhi Jane; Wang, Hong
    Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.
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    High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL
    (Elsevier, 2023) Kaur, Parminder; Lu, Xiaotong; Xu, Qi; Irvin, Elizabeth Marie; Pappas, Colette; Zhang, Hongshan; Finkelstein, Ilya J.; Shi, Zhubing; Tao, Yizhi Jane; Yu, Hongtao; Wang, Hong
    3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.
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    Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation
    (American Society for Microbiology, 2023) Ykema, Matthew; Ye, Kai; Xun, Meng; Harper, Justin; Betancourt-Solis, Miguel A.; Arias, Carlos F.; McNew, James A.; Tao, Yizhi Jane
    The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus–host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro.
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    Identification of a novel PRC2 recruiter in mammalian cells
    (2013-04-18) Gou, Yufeng; Ma, Jianpeng; Diehl, Michael R.; Tao, Yizhi Jane; Wang, Qinghua
    Polycomb repressive complex (PRC) 2 functions to repress thousands of target genes, and they are responsible for stem cell differentiation and carcinogenesis. However, how PRC2 are recruited to specific regions of their target genes remains elusive. In Drosophila, nine sequence-specific transcription factors including Zeste have been shown to act as PRC2 recruiters, but little is known about their homologues in mammalian cells, as a straightforward homology search failed to work in most cases. Aided by three-dimensional structure prediction and the use of the genomes of intermediate bridging species, we have identified a new protein, Zeste Homologue in humans (ZH), as the human homologue of Drosophila Zeste. Gel shift assays indicated that ZH binds to Zeste recognition sequence via its N-terminal DNA binding domain. ZH physically interacted with the components of PRC2 in GST-pull down assays. Chip-seq and Chip-qPCR experiments show the co-localization of ZH and PRC2 complex. Together, these findings revealed the critical function of ZH in recruiting PRC2 complexes to their target genes.
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    Initiation of RNA polymerization and polymerase encapsidation by a Picobirnavirus
    (2015-01-15) Collier, Aaron Michael; Tao, Yizhi Jane; Stewart, Charles R; Olson, John S; Prasad, B.V. V
    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How these RdRPs molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions that have highly variable answers depending on the virus family being examined. In this study, we have determined a 2.4 Å resolution structure of an RdRP from a human infecting strain of picobirnavirus (PBV). In addition to a conserved polymerase fold, the PBV RdRP possesses a unique, highly flexible 24-aa loop structure (aa495-518) located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays have shown that the wild-type RdRP is capable of initiating RNA synthesis using a de novo mechanism, while a mutant RdRP lacking the loop structure could only synthesize RNA through back-priming, suggesting that the loop likely functions as a platform for the priming nucleotide to bind. Unexpectedly, co-expression of the PBV RdRP with its respective capsid protein (CP) indicated that the PBV RdRP could not be incorporated into recombinant capsids in the absence of the viral genome. Additionally, the PBV RdRP exhibited a high affinity towards the conserved 5’-terminal sequence of the viral genome, suggesting that PBV RdRP molecules are likely packaged through their specific binding to viral RNA during assembly.
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    Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus
    (Public Library of Science, 2016) Collier, Aaron M.; Lyytinen, Outi L.; Guo, Yusong R.; Toh, Yukimatsu; Poranen, Minna M.; Tao, Yizhi Jane
    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.
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    KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase
    (Springer Nature, 2017) Wang, Yugang; Guo, Yusong R.; Liu, Ke; Yin, Zheng; Liu, Rui; Xia, Yan; Tan, Lin; Yang, Peiying; Lee, Jong-Ho; Li, Xin-jian; Hawke, David; Zheng, Yanhua; Qian, Xu; Lyu, Jianxin; He, Jie; Xing, Dongming; Tao, Yizhi Jane; Lu, Zhimin
    Histone modifications, such as the frequently occurring lysine succinylation1,2, are central to the regulation of chromatin-based processes. However, the mechanism and functional consequences of histone succinylation are unknown. Here we show that the α-ketoglutarate dehydrogenase (α-KGDH) complex is localized in the nucleus in human cell lines and binds to lysine acetyltransferase 2A (KAT2A, also known as GCN5) in the promoter regions of genes. We show that succinyl-coenzyme A (succinyl-CoA) binds to KAT2A. The crystal structure of the catalytic domain of KAT2A in complex with succinyl-CoA at 2.3 Å resolution shows that succinyl-CoA binds to a deep cleft of KAT2A with the succinyl moiety pointing towards the end of a flexible loop 3, which adopts different structural conformations in succinyl-CoA-bound and acetyl-CoA-bound forms. Site-directed mutagenesis indicates that tyrosine 645 in this loop has an important role in the selective binding of succinyl-CoA over acetyl-CoA. KAT2A acts as a succinyltransferase and succinylates histone H3 on lysine 79, with a maximum frequency around the transcription start sites of genes. Preventing the α-KGDH complex from entering the nucleus, or expression of KAT2A(Tyr645Ala), reduces gene expression and inhibits tumour cell proliferation and tumour growth. These findings reveal an important mechanism of histone modification and demonstrate that local generation of succinyl-CoA by the nuclear α-KGDH complex coupled with the succinyltransferase activity of KAT2A is instrumental in histone succinylation, tumour cell proliferation, and tumour development.
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    Molecular Basis of KAT2A Selecting Acyl-CoA Cofactors for Histone Modifications
    (AAAS, 2023) Li, Sha; Li, Nan; He, Jie; Zhou, Runxin; Lu, Zhimin; Tao, Yizhi Jane; Guo, Yusong R.; Wang, Yugang
    Emerging discoveries about undocumented acyltransferase activities of known histone acetyltransferases (HATs) advance our understandings in the regulation of histone modifications. However, the molecular basis of HATs selecting acyl coenzyme A (acyl-CoA) substrates for histone modification is less known. We here report that lysine acetyltransferase 2A (KAT2A) as an illustrative instance of HATs can selectively utilize acetyl-CoA, propionyl-CoA, butyryl-CoA, and succinyl-CoA to directly deposit 18 histone acylation hallmarks in nucleosome. By analyzing the co-crystal structures of the catalytic domain of KAT2A in complex with acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, succinyl-CoA, and glutaryl-CoA, we conclude that the alternative substrate-binding pocket of KAT2A and the length and electrostatic features of the acyl chain cooperatively determine the selection of the acyl-CoA substrates by KAT2A. This study reveals the molecular basis underlying the pluripotency of HATs that selectively install acylation hallmarks in nucleosomes, which might serve as instrumental mechanism to precisely regulate histone acylation profiles in cells.
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    Orsay Virus CP-δ Adopts a Novel β-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber
    (American Society for Microbiology, 2020) Guo, Yusong R.; Fan, Yanlin; Zhou, Ying; Jin, Miao; Zhang, Jim L.; Jiang, Hongbing; Holt, Matthew V.; Wang, Tao; Young, Nicolas L.; Wang, David; Zhong, Weiwei; Tao, Yizhi Jane
    Fiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematode Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ(1–101) (a deletion mutant containing the first 101 amino acid [aa] residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple β-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. The δ proteins from two other nematode viruses, Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.