Unlocking the PIP-box: A peptide library reveals interactions that drive high affinity binding to human PCNA.

The human sliding clamp Proliferating Cell Nuclear Antigen (hPCNA) interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine tune downstream processes. Here we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications, and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterised as KD values determined by surface plasmon resonance (SPR), spanned a 4000-fold range, from 1.83 nM to 7.59 μM. The hPCNA-bound peptide structures determined by X-ray crystallography and modelled computationally revealed inter- and intramolecular interaction networks which correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA-affinity to regulate DNA replication and repair processes. Additionally, these insights will be useful to future design of hPCNA inhibitors.