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

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 Borcherds 1,2$ , François-Xavier Theillet 3$ , Andrea Katzer 4$ , Ana Finzel 4 , Katie M. Mishall 1,2 , Anne Powell 1,2 , Hongwei Wu 1,2 , Wanda Manieri 5 , Christoph Dieterich 6 , Philipp Selenko 3 , Alexander Loewer 4 and Gary W. Daughdrill 1,2 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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