Abstract
Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m
7GTP. We report a binding affinity of 43.3
±
0.1 nM at 25 °C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m
7GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with
TΔ
S° contributing 15% to the overall value of Δ
G°. This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the Δ
G° for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein–protein interaction with a
K
d of 108.4
±
0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m
7GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.