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

Disordered Motifs and Domains in Cell Control

Poster Session II

40-POS Board 16 Phosphorylation Downstream of the PIP-Box Motif in Spd1 Regulates Association with PCNA Sebastian S. Broendum 1 , Noah Kassem 1 , Olaf Nielsen 3 , Kasper D. Rand 2 , Birthe B. Kragelund 1 . 1 University of Copenhagen,Department of Biology, SBiNLab, Copenhagen, Denmark, 2 University of Copenhagen, Department of Pharmacy, Biomacromolecular Analysis Laboratory, Copenhagen, Denmark, 3 University of Copenhagen, Department of Biology, Functional Genomics, Copenhagen, Denmark. Ribonucleotide Reductase (RNR) is ubiquitously involved in controlling the rate of mutation in cells and understanding its regulation is very important to improve our understanding of cancer and how it develops. In S. pombe, one way by which RNR is regulated is by a small intrinsically disordered inhibitory protein called Spd1. During DNA replication, Spd1 is degraded to allow synthesis of dNTPs. A key step in Spd1 degradation is the interaction with Proliferating Cell Nuclear Antigen (PCNA). Spd1 contains a short linear motif known as the PCNA interacting protein-box (PIP-box). For many PCNA-binding proteins, this interaction is mediated via their PIP-Box motif and mutation studies have confirmed that the PIP-Box in Spd1 is important for its interaction with PCNA. To resolve how this motif in Spd1 mediates the interaction with PCNA, we have structurally characterized Spd1 and the interaction between Spd1 and PCNA using Nuclear Magnetic Resonance spectroscopy and hydrogen-to-deuterium exchange mass spectrometry. We show that the Spd1 PIP-Box forms a transient α-helix in the unbound state and phosphorylation of Spd1 downstream of the PIP-Box destabilizes this α-helix. Phosphorylation also abolishes the interaction between Spd1 and PCNA and therefore we propose a model where phosphorylation of Spd1 regulates the interaction with PCNA. Furthermore, we discuss the role of the altered α-helix propensity in this process. Regulation of Spd1 degradation allows the cell to control RNR activity and thereby maintain a balanced level of dNTPs to minimize the rate of mutation in the cell.

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