Disordered Motifs and Domains in Cell Control - October 11-15, 2014 - page 37

Disordered Motifs and Domains in Cell Control Monday Speaker Abstracts
Riding with a Ubiquitin Ticket
Kylie Walters.
NCI, Frederick, Maryland, USA.
The compact 76 amino acid protein ubiquitin is used to signal for a broad spectrum of cellular
events. This modification is diversified by expansion into a ubiquitin polymer, formed through
eight possible linkages. Ubiquitin receptors contribute to determining the outcome of
ubiquitination by their specificity for distinct ubiquitin polymers. This talk will present new
interactions involving ubiquitin receptors and ubiquitin polymers, as well as functional
implications. By using NMR spectroscopy, we have found protein dynamics and disorder to play
distinct and defining functional roles.
Disorder and Residual Helicity Alter p53-Mdm2 Binding Affinity and Signaling in Cells
Wade Borcherds1,2$, François-Xavier Theillet 3$, Andrea Katzer4$, Ana Finzel4, Katie M.
Mishall1,2, Anne Powell1,2, Hongwei Wu1,2, Wanda Manieri5, Christoph Dieterich6, Philipp
Selenko3, Alexander Loewer4 and Gary W. Daughdrill1,2
1 Department of Cell Biology, Microbiology, and Molecular Biology, University of South
Florida, USA; 2 Center for Drug Discovery and Innovation, University of South Florida, Tampa,
FL, USA; 3 Department of NMR-supported Structural Biology, Leibniz Institute of Molecular
Pharmacology (FMP Berlin), 13125 Berlin, Germany; 4 Berlin Institute for Medical Systems
Biology, Max-Delbrueck-Center, 13125 Berlin, Germany; 5 Drug Discovery Department, Moffitt
Cancer Center, University of South Florida, FL, USA; 6 Max Planck Institute for Biology of
Ageing, 50931 Cologne, German
The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain
that undergoes coupled folding and binding when it interacts with partner proteins like the E3
ubiquitin ligase, Mdm2, and the 70 kDa subunit of replication protein A, RPA70. The secondary
structure and dynamics of six closely related mammalian orthologues of p53TAD were
investigated using nuclear magnetic resonance (NMR) spectroscopy. Clustering analysis showed
that the divergence in transient helical secondary structure of the p53TAD orthologues is more
extensive than the amino acid sequence divergence. In contrast, strong correlations were
observed between the backbone dynamics of the orthologues and the sequence identity matrix,
suggesting that the dynamic behavior of IDPs is under positive evolutionary selection. Mutating
conserved prolines that flank the Mdm2 binding site to Alanines doubled the level of transient
helical secondary structure in this region. This doubling of transient helical secondary structure
increased the in vitro binding affinity between p53TAD and Mdm2. The in vivo binding affinity
between full-length p53 and Mdm2 was also increased in the proline mutants. This increase in
binding affinity disrupted the expression of p53 target genes and inhibited the ability of cells to
arrest in G1 following radiation induced DNA damage. Taken together our results demonstrate
that the transient helical secondary structure of p53TAD has been finely tuned by evolution and
disrupting this structure has deleterious effects on target gene expression and cell fate decisions.
This research was supported by the Deutsche Forschungsgemeinschaft (Emmy Noether grant
PS1794/1-1 to PS), the Association pour la Recherche contre le Cancer (postdoctoral fellowship
to FXT), the European Union FP7 (Marie Curie CIG to AL), the American Cancer Society
(RSG-07-289-01-GMC to GWD) and the National Science Foundation (MCB-0939014 to
GWD).
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