PAPOAC and FIP1 compete for binding to the PAPOA RNA-binding domain. (A) Domain organization of human PAPOA. The truncated constructs used here are indicated in orange (PAPOA structured core) or yellow (PAPOAC). (B) Overlay of 25 computationally predicted models of the PAPOA1–513–PAPOAC complex shown in two orientations (PAPOAC residues 720–729 are not shown for clarity). The PAPOA structured domains and active sites are indicated. (C) Close-up of the putative binding interface. Labeled residues of the PAPOA core were changed to glutamic acid. (D) Coomassie-stained SDS-PAGE analysis of pull-down experiment showing that negatively charged PAPOA mutations in the predicted PAPOA–PAPOAC interface disrupt the interaction. (E) Coomassie-stained SDS-PAGE analysis of competition pull-down experiment showing that PAPOAC and FIP1 compete for the same binding surface on the PAPOA structured core. (F) Quantification of the competition pull-down experiment performed in triplicates.
