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 ca talytically 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 subs titutions of the active center cleft residues significantly impair the depu rinating and ribosome inhibitory activity of PAP, Here we employed site-dir ected mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance tech nology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha -sarcin/ricin stem loop of rRNk Our findings p resented herein provide experimental evidence that besides the catalytic si te, 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 coul d, via electrostatic interactions, contribute to both the correct orientati on and stable binding of the substrate RNA molecules in PAP active site poc ket. The insights gained from this study also explain why and how the conse rved charged and polar side chains located at the active center cleft of PA P and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the c atalytic removal of the adenine base from target rRNA substrates by affecti ng the binding interactions between PAP and rRNA.