A large network of proteins is responsible for replicating DNA with high fidelity and for repairing DNA damage through different pathways. PCNA is a global hub in DNA metabolism that interacts with a large number of proteins involved in a variety of DNA-related processes [34]. As PCNA is a symmetric homotrimer in solution, it has three identical hydrophobic pockets to simultaneously bind different partners and coordinate a variety of functions in space and time [35]. Most of the proteins that bind PCNA either are IDPs (intrinsically disordered proteins) or have IDRs (intrinsically disordered regions). IDPs lack defined secondary and tertiary structures under physiological conditions. Although they fulfil important biological functions across all domains of life, they are more abundant in eukaryotes [36]. In particular, most transcription factors, as well as proteins involved in signal transduction, in eukaryotic organisms are predicted to be disordered or to contain disordered regions [37]. This reveals a correlation between complex cell regulation and the greater presence of IDPs/IDRs [38]. Moreover, most of the proteins associated with cancer have been identified as IDP or IDR-containing proteins (79%), which underlines the crucial roles they play in several cellular events that are altered in cancer, such as cell proliferation, DNA repair, and apoptosis [39]. The evolutionary advantage of IDPs likely lies in their plasticity that allows them to interact with many ligands and their vast regulation through post-translational modifications [40]. Disordered proteins have large accessible surface areas, which increase their ability to interact with diverse binding partners through short linear motifs (SLiMs) [41]. Many of the proteins that interact with PCNA display a characteristic SLiM known as PIP box (PCNA interacting protein-box) or an extended version called PIP degron. The canonical PIP box motif is QXXhXXaa, where h is an aliphatic hydrophobic residue (frequently I, L, or M) and a is an aromatic hydrophobic one (F, W, or Y), whereas X can be any amino acid [9]. The PIP degron motif targets PCNA for degradation and also harbors a basic residue (K or R) four amino acids after the second aromatic residue, as well as a TD motif just before the aromatic residues [42]. Additionally, a novel motif was identified and found to be present in several PCNA interacting proteins, named KA box, that consists of residues K-A-(A/L/I)-(A/L/Q)-x-x-(L/V) [43,44].

Structural studies of PIP box containing proteins or derived peptides in complex with human PCNA have unveiled the molecular details of the PIP motif-PCNA interface. The crystal structure of a p21 fragment bound to human PCNA was the first structural characterization of this interface (PDB code: 1AXC) [22]. Since then, several co-crystal structures of PCNA with different ligands have been solved. Overall, all PCNA interacting proteins adopt a similar conformation, which consists of an extended N-terminal region, a 3₁₀ helical turn of four residues enclosed by the hydrophobic residues of the PIP box, and a C-terminal region of variable length that sometimes adopts a β strand secondary structure and interacts with the IDCL. The conserved helix inserts into the hydrophobic pocket of PCNA, whereas the glutamine sticks into the so-called Q-pocket, establishing hydrogen bonds with the backbone of PCNA [22,42]. The assignment of the human PCNA NMR spectrum [24] allowed the analysis of the ¹H-¹⁵N correlation spectra of PCNA in the presence of different ligands and the calculation of the perturbations in the NMR signals. Thereby, the NMR data provided complementary or new information regarding these interactions in solution at the residue level [45].

The available structural information of PCNA in complex with different partners reveals that three ligands are able to bind simultaneously the three identical protomers of the PCNA ring. Isothermal titration calorimetry (ITC) data with different peptides are all well fitted with a model assuming one set of equivalent sites, with no evidence of binding cooperativity. Therefore, it seems that ligands compete for binding with PCNA based on their affinities, which can be modulated by post-translational modifications [9].

The intrinsically disordered protein p21 is one of the PCNA binding partners showing the highest affinity (Table 1), probably because it needs to displace other proteins to block replication in response to DNA damage. This high affinity relies on efficient hydrophobic packing, as well as electrostatic interactions with the C-terminal region of PCNA [46]. Studies with variable length peptides of p21 suggest that basic residues at the N and C-termini encompassing the PIP box protein also contribute to an increase in the binding affinity. A recent study combining experimental and computational approaches confirmed this correlation between positive patches of residues flanking the PIP box motif and a strong affinity for PCNA [41]. Moreover, FEN1 endonuclease and p68 (also known as p66, the third subunit of Pol δ) exhibit fewer basic residues than p21, and accordingly present lower affinity (Table 1). However, the comparison of the structure of PCNA with full length FEN1 and with a 20-residue long PIP fragment reveals additional contacts involving the core domain and the C-terminal region of FEN1, which increase the affinity of FEN1 by three orders of magnitude.