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 K D 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 K D 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.Mdm2 and MdmX (also known as Mdm4) are arguably the most important regulators of p53, being responsible for generating the dynamic characteristics of the p53 signaling pathway (1). Mdm2 is a classic p53 target gene and it was recently shown that p53 also induces MdmX transcription (2,3). Both Mdm2 and MdmX form negative feedback loops that regulate p53 activity using different mechanisms and mouse models show that Mdm2 and MdmX are necessary for controlling p53 activity (4-9). Mdm2 and MdmX contain N-terminal domains that bind tightly to residues 17-29 of the p53 transcriptional activation domain (p53TAD). MdmX also contains a short linear binding motif between residues 195-206 that resembles residues 17-29 of p53TAD. This motif interacts with the p53 binding domain (p53BD) of MdmX and interferes with p53 binding (10,11). We recently showed that a short peptide containing MdmX residues 195-206 binds to the p53BD of MdmX with a K D ¼ 8.3 5 0.1 mM (11). Based on this previous work, we hypothesize the intramolecular binding motif at residues 195-206, referred to as the WW motif because it contains two tryptophan residues, is connected to the p53-binding domain of MdmX by a long flexible linker that controls the intramolecular binding affinity. This hypothesis is supported by the IUPred disorder prediction of human MdmX shown in Fig. 1 a (12). IUPred correctly predicts the p53 binding domain of MdmX is ordered (13,14). After the p53 binding domain is a long segment from residues 112-181 predicted to be disordered, and a short segment from residues 182-211, which includes the WW motif, with disorder tendency values that dip below 0.5. Previous studies have shown that short protein segments with a disorder tendency <0.5 will often correspond to linear binding motifs (15)(16)(17). In the case of MdmX, the binding of this linear motif is intramolecular. It is also worth noting that ANCHOR correctly predicts the WW motif is a protein bindi...

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

“…have mostly been used in efforts to optimize multivalent drugs through avidity (15,16). In molecular biology, effective concentrations are rarely measured experimentally, but usually estimated theoretically from the volume in which the tethered ligand is free to diffuse (17)(18)(19)(20)(21). While such simple geometric models are commonly used, they have not been tested in complex biological systems.…”

Conformational landscape of full-length Smad proteins

“…The linker appears to act like a mechanical element, allowing the approximation and separation of the MH1 and MH2 domains without retaining them through a long and stable compact interaction. These transitions between compact and open states are usually favored for many multidomain DNA-binding proteins (37,60,61), and in the case of Smad proteins, the expanded states could facilitate linker modification and cofactor association by providing ensembles of conformations separated by almost no energy barriers. Moreover, we observed that Smad4 is mostly monomeric (and not a trimer, as observed in crystals of MH2 domains) whereas Smad2 populates an equilibrium containing monomers, dimers and trimers, with the trimers being less abundant in full-length proteins than in the context of isolated MH2 domains.…”

Linker dependence of avidity in multivalent interactions between disordered proteins

“…The system is artificial, but instead estimated from polymer models. [29][30][31][32][33] The effective concentrations are sensitive to the linker sequence, 15 so without direct measurement of the effective concentrations it would be difficult to detect deviations from theoretical predictions. We thus believe that the primary strength of the present study is the ability to critically assess theoretical models of avidity in dynamic multidomain proteins.…”