Active Center Cleft Residues of Pokeweed Antiviral Protein Mediate Its High-Affinity Binding to the Ribosomal Protein L3

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• 作者：Francis Rajamohan ; Zahide Ozer ; Chen Mao ; and Fatih M. Uckun
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 7, 2001
• 年：2001
• 卷：40
• 期：31
• 页码：9104 - 9114
• 全文大小：777K
• 年卷期：v.40,no.31(August 7, 2001)
• ISSN：1520-4995

文摘

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP) which catalyticallycleaves a specific adenine base from the highly conserved -sarcin/ricin loop (SRL) of the large ribosomalRNA and thereby inhibits the protein synthesis. The ribosomal protein L3, a highly conserved proteinlocated at the peptidyltransferase center of the ribosomes, is involved in binding of PAP to ribosomes andsubsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants withalanine substitution of the active center cleft residues ⁶⁹NN⁷⁰ (FLP-4) and ⁹⁰FND⁹² (FLP-7) that are notdirectly involved in the catalytic depurination at the active site exhibit >150-fold reduced ribosomeinhibitory activity [(2000) J. Biol. Chem. 275, 3382-3390]. We hypothesized that the partially exposedhalf of the active site cleft could be the potential docking site for the L3 molecule. Our modeling studiespresented 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 rRNAand 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 thatthese mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins forribosomes and recombinant L3 protein were calculated from rate constants and analysis of binding usingsurface plasmon resonance biosensor technology. Here, we show that, compared to wild-type PAP, FLP-4/⁶⁹AA⁷⁰ and FLP-7/⁹⁰AAA⁹² exhibit significantly impaired affinity for ribosomes and L3 protein, whichmay account for their inability to efficiently inactivate ribosomes. By comparison, recombinant PAP mutantswith alanine substitutions of residues ²⁸KD²⁹ and ¹¹¹SR¹¹² that are distant from the active center cleftshowed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP withalanine substitution of the active center cleft residues N69 (FLP-20), F90 (FLP-21), N91 (FLP-22), orD92 (FLP-23) also showed reduced ribosome binding as well as reduced L3 binding, further confirmingthe importance of the active center cleft for the PAP-ribosome and PAP-L3 interactions. The experimentalfindings presented in this report provide unprecedented evidence that the active center cleft of PAP isimportant for its in vitro binding to ribosomes via the L3 protein.