Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

Abstract

The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAPmediated depurination of ribosomal RNA.

Transcription Factor STAT5A Is a Substrate of Bruton's Tyrosine Kinase in B Cells

“…Site-directed Mutagenesis-Point mutations were introduced into the stat5a cDNA by PCR (47). The codon for arginine 618 within the SH2 domain was mutated to a codon for lysine.…”

Structure-Based Design and Engineering of a Nontoxic Recombinant Pokeweed Antiviral Protein with Potent Anti-Human Immunodeficiency Virus Activity

“…We have tested recombinant PAP against a broad panel of viruses in vitro and documented that it is as active as native PAP against both DNA and RNA viruses (16,17). We were also able to determine the X-ray crystal structure of PAP at a 2.1-Å resolution (8, 9).More recently, we have used a molecular model of PAP-RNA interactions (19,21,22) for the rational design of PAP mutants with potent anti-HIV activities. In the present study, two such recombinant PAPs, FLP-102 ( 151 AA 152 ) and ), have been engineered, produced, and tested both in vitro and in vivo.…”

“…Molecular modeling studies for the rational design of recombinant PAPs were performed as previously described in detail (19,21,22). Recombinant wild-type PAP (phosphate-buffered saline [PBS]-PAP) was obtained by subcloning the PAP-I gene (amino acids 22 to 313) into the pBluescript SKϪ expression vector (17).…”

“…Five micrograms of E. coli 23S and 16S rRNAs (Roche Molecular Biochemicals) was incubated with increasing amounts of wild-type or mutant PAP in 50 l (final volume) of binding buffer (25 mM Tris-HCl [pH 7.8], 10 mM KCl, 5 mM MgCl 2 , 2% glycerol) at 37°C for 1 h. The rRNA was extracted with phenol-chloroform (24:24), precipitated with ethanol, and treated with 20 l of 1 M aniline acetate (pH 4.5) for 30 min on ice. The rRNA was precipitated with ethanol, electrophoresed in a 6% urea-polyacrylamide gel, and stained with ethidium bromide as described previously (19).…”

“…More recently, we have used a molecular model of PAP-RNA interactions (19,21,22) for the rational design of PAP mutants with potent anti-HIV activities. In the present study, two such recombinant PAPs, FLP-102 ( 151 AA 152 ) and FLP-105 ( 191 AA 192 ), have been engineered, produced, and tested both in vitro and in vivo.…”