Active center cleft residues of pokeweed antiviral protein mediate its high-affinity binding to the ribosomal protein L3

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP) w hich catalytically cleaves a specific adenine base from the highly conserve d alpha -sarcin/ricin loop (SRL) of the large ribosomal RNA and thereby inh ibits the protein synthesis. The ribosomal protein L3, a highly conserved p rotein located at the peptidyltransferase center of the ribosomes, is invol ved in binding of PAP to ribosomes and subsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants with alanine subst itution of the active center cleft residues (NN70)-N-69 (FLP-4) and (FND92) -F-90 (FLP-7) that are riot directly involved in the catalytic depurination at the active site exhibit > 150-fold reduced ribosome inhibitory activity [(2000) J. Biol. Chem: 275, 3382-3390]. We hypothesized that the partially exposed half of the active site cleft could be the potential docking site for the L3 molecule. Our modeling studies presented herein indicated that P AP residues 90-96, 69-70, and 118-120 potentially interact with L3. Therefo re, mutations of these residues were predicted to result in destabilization of interactions with rRNA and lead to a lower binding affinity with L3. In the present structure-function relationship study, coimmunoprecipitation a ssays with an in vitro synthesized yeast ribosomal protein L3 suggested tha t these mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins for ribosomes and recombinant L3 protein were c alculated from rate constants and analysis of binding using surface plasmon resonance biosensor technology. Here, we show that, compared to wild-type PAP, FLP-4/(69)AA(70) and FLP-7/(90) AAA(92) exhibit significantly impaired affinity for ribosomes and L3 protein, which may account for their inabili ty to efficiently inactivate ribosomes. By comparison, recombinant PAP muta nts with alanine substitutions of residues (KD29)-K-28 and (SR112)-S-111 th at are distant from the active center cleft showed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP with alan ine substitution of the active center cleft residues N69 (FLP-20), F90 (FLP -21), N91 (FLP-22), or D92 (FLP-23) also showed reduced ribosome binding as well as reduced L3 binding, further confirming the importance of the activ e center cleft for the PAP-ribosome and PAP-L3 interactions. The experiment al findings presented in this report provide unprecedented evidence that th e active center cleft of PAP is important for its in vitro binding to ribos omes via the L3 protein.