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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