Intrinsically unstructured domains of Arf and Hdm2 form bimolecular oligomeric structures in vitro and in vivo.
Arf, Hdm2, and p53 regulate the tumor-suppressor pathway that is most frequently disrupted in human cancer. In the absence of tumorigenic stress, Hdm2 actively attenuates p53-dependent cell cycle arrest and apoptosis by mediating ubiquitination-dependent degradation of p53. Mitogenic stress activates Arf, which indirectly activates p53 by binding to and nullifying the anti-p53 activities of Hdm2. Small conserved domains within Arf and Hdm2 mediate their direct interaction. Individually, these domains are intrinsically unstructured and, when combined in vitro, cofold into bimolecular oligomeric structures that resemble amyloid fibrils in some features. Detailed structural characterization of Hdm2/Arf complexes has previously been hampered by their heterogeneity and large size. Here, we report that a nine-residue fragment of the N-terminus of mouse Arf (termed "A1-mini") cofolds specifically with the Arf-binding domain of Hdm2 to form bimolecular oligomers. We characterized these unprecedented structures using analytical ultracentrifugation and NMR spectroscopy, providing insights into their structural organization. The A1-mini peptide not only binds specifically to Hdm2 in vitro but also recapitulates the nucleolar localization features of full-length Arf in cells. Furthermore, larger fragments of Arf that contain the A1-mini segment have previously been shown to activate p53 in mouse and human cells. Our studies provide the first insights into the molecular basis through which Arf nullifies the p53-inhibiting activity of Hdm2, indirectly activating the tumor-suppressor function of p53 in mammalian cells.
Sivakolundu
Sivashankar G
SG
Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
Nourse
Amanda
A
Moshiach
Simon
S
Bothner
Brian
B
Ashley
Chimere
C
Satumba
John
J
Lahti
Jill
J
Kriwacki
Richard W
RW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism; D017124:Conserved Sequence; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D013113:Spin Labels; D025803:Tumor Suppressor Protein p14ARF / Q000737:chemistry* / Q000378:metabolism
D013113:Spin Labels; D025803:Tumor Suppressor Protein p14ARF; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2