Optimal Affinity Enhancement by a Conserved Flexible Linker Controls p53 Mimicry in MdmX

Abstract

MdmX contains an intramolecular binding motif that mimics the binding of the p53 tumor suppressor. This intramolecular binding motif is connected to the p53 binding domain of MdmX by a conserved flexible linker that is 85 residues long. The sequence of this flexible linker has an identity of 51% based on multiple protein sequence alignments of 52 MdmX homologs. We used polymer statistics to estimate a global KD value for p53 binding to MdmX in the presence of the flexible linker and the intramolecular binding motif by assuming the flexible linker behaves as a wormlike chain. The global KD estimated from the wormlike chain modeling was nearly identical to the value measured using isothermal titration calorimetry. According to our calculations and measurements, the intramolecular binding motif reduces the apparent affinity of p53 for MdmX by a factor of 400. This study promotes a more quantitative understanding of the role that flexible linkers play in intramolecular binding and provides valuable information to further studies of cellular inhibition of the p53/MdmX interaction.