Dataset Information

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.

SUBMITTER: Borcherds W

PROVIDER: S-EPMC5443996 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Borcherds Wade W Becker Andreas A Chen Lihong L Chen Jiandong J Chemes Lucía B LB Daughdrill Gary W GW

Biophysical journal 20170506 10

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<sub>D</sub> value for p53 binding to MdmX in the presence of the flexible linker and t ...[more]

PMID: 28487147

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Project description:Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

Dataset Information

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

Publications

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

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