X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inacti vating proteins (RIP), which depurinate ribosomal RNA through their site-sp ecific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-c ytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 Angstrom resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving on ly the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivatin g proteins and its cleavage by PAP provides unprecedented evidence for a br oad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 Angstrom crystal struct ure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring i s strongly bound in the active site, forming four hydrogen bonds with activ e site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only t hree hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms on ly one hydrogen bond with the guanido group of Arg135.