Modeling and alanine scanning mutagenesis studies of recombinant pokeweed antiviral protein

Phytolacca americana-derived naturally occurring ribosome inhibitory protei n pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically r emoves a specific adenine residue from the stem loop of ribosomal RNA. We h ave employed molecular modeling studies using a novel model of PAP-RNA comp lexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA bindin g active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP, As anticipated, alanin e substitutions by site-directed mutagenesis of the PAP active site residue s Tyr(72), Tyr(123), Glu(176), and Arg(179) that directly participate in th e catalytic deadenylation of RNA resulted in greater than 3 logs of loss in depurinating and ribosome inhibitory activity. Similarly, alanine substitu tion of the conserved active site residue Trp(208), which results in the lo ss of stabilizing hydrophobic interactions with the ribose as well as a hyd rogen bond to the phosphate backbone of the RNA substrate, caused greater t han 3 logs of loss in enzymatic activity, By comparison, alanine substituti ons of residues (KD29)-K-28, (FE81)-F-80, (SR112)-S-111, (FL167)-F-166 that are distant from the active site did not significantly reduce the enzymati c activity of PAP. Our modeling studies predicted that the residues of the active center cleft could via electrostatic interactions contribute to both the correct orientation and stable binding of the substrate RNA molecule i n the active site pocket. Nota bly, alanine substitutions of the highly con served, charged, and polar residues of the active site cleft including (48) Ky(49), (RR68)-R-67, (VN70)-V-69, and (FND92)-F-90 substantially reduced th e depurinating and ribosome inhibitory activity of PAP, These results provi de unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cle ft also play a critical role in the PAP-mediated depurination of ribosomal RNA.