TY - JOUR
T1 - The antiprion compound 6-aminophenanthridine inhibits the protein folding activity of the ribosome by direct competition
AU - Pang, Yanhong
AU - Kurella, Sriram
AU - Voisset, Cécile
AU - Samanta, Dibyendu
AU - Banerjee, Debapriya
AU - Schabe, Ariane
AU - Gupta, Chanchal Das
AU - Galons, Hervé
AU - Blondel, Marc
AU - Sanyal, Suparna
PY - 2013/6/28
Y1 - 2013/6/28
N2 - Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.
AB - Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.
UR - http://www.scopus.com/inward/record.url?scp=84879591681&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84879591681&partnerID=8YFLogxK
U2 - 10.1074/jbc.M113.466748
DO - 10.1074/jbc.M113.466748
M3 - Article
C2 - 23673663
AN - SCOPUS:84879591681
SN - 0021-9258
VL - 288
SP - 19081
EP - 19089
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -