Structural insights into the CP312R protein of the African swine fever virus.

African swine fever (ASF) is a lethal hemorrhagic disease that affects domestic pigs and wild boars. There is no medication available for ASF to date. The ability to mount antigen-specific responses to viral vectored CP312R makes it a crucial potential target for designing vaccines or drugs. This study determined the crystal structure of ASFV CP312R at 2.32 Å and found it to be a monomer with a single-stranded DNA binding core domain with a clear five-strands ß-barrel OB-fold architecture. Electrophoretic mobility shift assay and size-exclusion chromatography characterization assay further confirmed the single-stranded DNA (ssDNA)-binding property of ASFV CP312R. This study revealed the structure and preliminary ssDNA interaction mechanisms of ASFV CP312R, providing new clues for developing new antiviral strategies.

Crystal Structure of the Disease-Specific Protein of Rice Stripe Virus.

The rice stripe virus (RSV) is responsible for devastating effects in East Asian rice-producing areas. The disease-specific protein (SP) level in rice plants determines the severity of RSV symptoms. Isothermal titration calorimetry (ITC) and bimolecular fluorescence complementation (BiFC) assays confirmed the interaction between an R3H domain-containing host factor, OsR3H3, and RSV SP in vitro and in vivo. This study determined the crystal structure of SP at 1.71 Å. It is a monomer with a clear shallow groove to accommodate host factors. Docking OsR3H3 into the groove generates an SP/OsR3H3 complex, which provides insights into the protein-binding mechanism of SP. Furthermore, SP's protein-binding properties and model-defined recognition residues were assessed using mutagenesis, ITC, and BiFC assays. This study revealed the structure and preliminary protein interaction mechanisms of RSV SP, shedding light on the molecular mechanism underlying the development of RSV infection symptoms.

Structure-guided rational design of the Geobacillus thermoglucosidasius feruloyl esterase GthFAE to improve its thermostability.

Feruloyl esterases are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links. GthFAE from Geobacillus thermoglucosidasius was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing. To improve the enzymatic properties rationally and efficiently, the structure of GthFAE was solved at 1.9 Å, revealing a core domain of classical α/ß hydrolase fold and an inserted α/ß cap domain. In silico analysis based on it helped us to investigate whether the residues at the active center have positive effects on the stability, and how. Several site-directed mutations were conducted, of which substitutions at residues T41 and T150 apparently improved the thermostability. The combination mutant T41N/T150R exhibited an optimal temperature of 65 °C, a 6.4 °C higher Tm compared to wild type by 80 °C, and a 35-fold longer in half-life (201 min) at 70 °C. Molecular dynamics simulations further illustrated that the structure of T41N/T150R was more stable than the wild type and T150R stabilized the cap domain by introducing salt bridges to the region with E154 and D164. This study not only highlighted residues within the active center on their thermostability improving effects, but also contributed to the prospective industrial application of GthFAE.