Yun-Sang Tang

VVA2 (volvatoxin A chain 2) is a cardiotoxic protein purified from Volvariella volvacea. Its biological activities include hemolysis, writhing reaction, neurotoxicity, and ventricular systolic arresting activity. The cytotoxicity of VVA2 was mainly considered due to its pore-forming activity. Here we report a novel biological activity of its variants VVA2 I82E/K86K as a duplex-specific nuclease. Recombinant VVA2 variant I82E/L86K (Re-VVA2 I82E/L86K), deprived of the oligomerization property, shows increased nuclease activity compared to VVA2. Re-VVA2 I82E/L86K converts supercoiled DNA (Replicative form I, RF I) into nicked form (RF II) and linear form (RF III) in the presence of Mg²⁺ or Mn²⁺. Besides plasmid DNA, it also exhibits nuclease activity on E. coli genomic DNA rather than ssDNA or RNA. Re-VVA2 I82E/L86K preferentially cleaves dG-dC-rich dsDNA regions and shows the best performance at pH 6–9 and 55 °C. Our structure–function study has revealed amino acid E111 may take an active part in nuclease activity through interacting with metal ions. Based on the sequences of its cleavage sites, a “double-hit” mechanism was thereby proposed. Given that Re-VVA2 I82E/L86K did not exhibit the conserved nuclease structure and sequence, it is considered an atypical duplex-specific nuclease.

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.