MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

Abstract

Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2–MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2–MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (K_D) in the micromolar range for the MDM2–MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2–MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2–MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation.

Keywords:

• MDM2
• MDM4
• atomic force spectroscopy
• surface plasmon resonance

Acknowledgments

This work was supported by the Italian Association for Cancer Research (AIRC): Grant IG15866 (SC) and Grant IG12767 (FM).

Ethical approval was not required by the institutional review boards of Università della Tuscia, Università Cattolica di Roma, or Consiglio Nazionale delle Ricerche (CNR) because the study used safe purified proteins for in vitro interaction studies.

Disclosure

The authors report no conflicts of interest in this work.