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) which catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA and thereby inhibits the protein synthesis. The ribosomal protein L3, a highly conserved protein located at the peptidyltransferase center of the ribosomes, is involved in binding of PAP to ribosomes and subsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants with alanine substitution of the active center cleft residues (69)NN(70) (FLP-4) and (90)FND(92) (FLP-7) that are not 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 PAP residues 90--96, 69--70, and 118--120 potentially interact with L3. Therefore, 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 assays with an in vitro synthesized yeast ribosomal protein L3 suggested that these mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins for ribosomes and recombinant L3 protein were calculated 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 inability to efficiently inactivate ribosomes. By comparison, recombinant PAP mutants with alanine substitutions of residues (28)KD(29) and (111)SR(112) that are distant from the active center cleft showed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP with alanine 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 active center cleft for the PAP--ribosome and PAP--L3 interactions. The experimental findings presented in this report provide unprecedented evidence that the active center cleft of PAP is important for its in vitro binding to ribosomes via the L3 protein.
Rajamohan
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Biotherapy Program, Parker Hughes Cancer Center, Parker Hughes Institute, St. Paul, Minnesota 55113, USA. frajamohan@ih.org
Ozer
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Mao
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Uckun
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D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry / Q000378:metabolism; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000378:metabolism*; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D010940:Plant Proteins; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein