Extreme multivalency and a composite short linear motif facilitate PCNA-binding, localisation and abundance of p21 (CDKN1A).

Cyclin-dependent kinase inhibitor 1 (CDKN1A; also known as p21) promotes cell cycle arrest and regulates DNA replication and DNA repair by high-affinity binding to proliferating cell nuclear antigen (PCNA) using a C-terminal short linear motif (SLiM). High-affinity binding to PCNA is driven by positively charged flanking regions of the SLiM, but the molecular details of their interaction as well as their roles for other p21 functions are not known. Using biophysics to study the interaction between PCNA and p21 variants with different Lys/Arg compositions in the flanking regions, as well as using D-amino acids, we find that the flanking regions of p21 bind to PCNA likely through an interaction driven by complementary charges without specific contacts. Although the exact Lys/Arg composition of the p21 flanking regions is unimportant for high-affinity PCNA binding, these positions are conserved in p21 orthologs, implying a conserved biological function. Accordingly, in cell-based experiments, we find that, while the flanking regions affect p21 abundance, both the context and the Lys/Arg composition of the N-terminal flanking region are crucial for p21 nuclear localisation. Such integration of SLiMs into a composite SLiM may be a widespread phenomenon and complicates the separation of function and drug development.