Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA.
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which 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 molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.
Rajamohan
F
F
Biotherapy Program, Parker Hughes Cancer Center, and the Departments of Protein Engineering, Structural Biology, and Virology, Parker Hughes Institute, St. Paul, Minnesota 55113, USA. frajamohan@ih.org
Mao
C
C
Uckun
F M
FM
D000818:Animals; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D020349:Surface Plasmon Resonance
D005656:Fungal Proteins; D010940:Plant Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C014801:alpha-sarcin; D004722:Endoribonucleases; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein