Mdm2 and MdmX share high structural similarity in their N-terminal domains, yet dual inhibitors are challenging to design due to differences in the conformations of the binding pockets, and notably of the proposed gatekeeper residue, Y100/99. Analysis of crystal structures and molecular dynamics (MD) simulations of complexes of Mdm2 and MdmX resulted in the identification of a water molecule with a long residence time that appears to be modulated by the conformation of Y100/99. These observations lead us to speculate that dual inhibitors either (i) stabilize both Mdm2 and MdmX with Y100/99 in the open conformation typically seen in complexes of Mdm2 with p53, or (ii) the dual inhibitors are agnostic to the conformation of Y100/99. The recently developed potent dual inhibitory stapled peptide Atsp7041 appears to be agnostic to the conformation of the gatekeeper residue. Proteins 2017; 85:1493-1506. © 2017 Wiley Periodicals, Inc.

loop. HTS-EQ involves competitive binding of hnRNP A1 to a completely randomized pool of ESS3 apical loop variants (n=16,384). Quantitative analysis of the resulting affinity distribution reveals RNA sequence; motif copy number; motif spacing; and secondary structure determine specificity by modulating rates of productive hnRNP A1-RNA encounters. To investigate if these rules can be extended to other viral systems, we determined a hybrid SAXS/NMR structure of hnRNP A1 in complex with an IRES element from Enterovirus 71. Indeed, the structure and biophysical studies of the complex reveal a common set of mechanistic principles determine functional hnRNP A1-RNA interactions. Thus, our work provides significant insights into the combinatorial factors that determine how viral RNA elements recruit host RNA binding proteins to control gene expression.