Molecular basis of the Arf and Hdm2 interaction.

摘要

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14 Arf stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14Arf are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition forming extended structures comprised of β-strands. Domains from both proteins are necessary and sufficient for the formation of the highly stable extended β structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of 5 amino acids using surface plasmon resonance (SPR). SPR and circular dichroism (CD) spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14Arf (human) and p19Arf (mouse) interact with Hdm2 through two short motifs present in their N-termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235–264 and 270–289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains responsible provide opportunities for the development of anticancer therapeutics. Analysis of the β-assemblies, using electron microscopy, showed that extended fibrils were formed by short peptides containing the high affinity binding site. This is the first report on β-fibrils in which domains from two proteins are involved in the formation of extended structures and may provide a platform for the assembly of macromolecular complexes useful in a wide range of nanotechnologies.