FC0088
PCNA-associated factor, p15^PAF  -  PCNA

Biological function
p15^PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp.

Domain organization/sequence features
Many PCNA-interacting proteins bind through the PIP-box, a consensus sequence with the pattern QXXhXXaa, where h is an aliphatic-hydrophobic residue, a is an aromatic-hydrophobic and X is any residue. The recognition surface on PCNA is small, contacting 11–18 residues of the ligand. A common feature of the PIP-box region in these proteins is its disordered nature, providing an adaptable, yet specific contact site whose affinity for PCNA can be tuned by subtle changes in the sequence. The regions flanking these tightly bound residues are usually not visible in electron density maps, suggesting that the chain breaks away from the clamp surface.

Structural evidence
The central region of p15, including the PIP-box, is tightly bound to the front-face and inner surface of PCNA, while the N- and C-termini remain predominantly disordered at its back- and front-face, respectively, with transient interactions at the PCNA outer surface. NMR signal intensities decrease generally and most notably for the central residues V53-D75 including the PIP box (⁶²QKGIGEFF⁶⁹). The p15 NMR signals still visible in the complex retain their low dispersion, indicating that they remain largely disordered and flexible. NMR spectra suggests only transient interactions between the p15 termini and PCNA. In the ensemble of p15–PCNA complexes, most of the bound p15 chains have their flexible N- and C-termini protrude at the opposite back and front faces of the PCNA ring, respectively. In structure determined from X-ray crystallography, only residues 51-71 are visible in the electron density map, but the side chains of N51, R70 and L71 could not be reliably modelled.

Biochemical evidence
Changes in the NMR spectra of p15 and PCNA in the presence of each other confirm a direct interaction between both proteins. Three p15 molecules bind to the trimeric PCNA ring with low micromolar affinity (1.1 μM), with no indication of cooperativity. Truncation constructs show that the affinity of p15^50-77 is reduced by a factor of 5, while that of p15^59-70 is reduced by 33-fold. The N-terminal regions (2-31 AA) provides small contribution to catalysis by transient interactions. This indicates that the disordered 71-77 region importantly contributes to binding.

Structure/Mechanism
The central region of p15, including the PIP-box, is tightly bound to the front-face and inner surface of PCNA, while the N- and C-termini remain predominantly disordered at its back- and front-face, respectively, with transient interactions at the PCNA outer surface. NMR titration is consistent with a binding mode in which the full-length p15 N terminus passes though the PCNA ring. p15 could also be degraded by the 20S proteasome independent of ubiquitin and ATP, most likely due to its intrinsically disordered state. This degradation is strongly and specifically inhibited in the presence of PCNA, despite most of the p15 chain remaining flexible and disordered in the bound form. p15 can only be degraded when dissociated from PCNA. This reduction indicates that p15 is less accessible to the proteasome even when dissociating from PCNA, possibly because part of it remains trapped within the PCNA ring.

Mechanism category
tethering

Significance
Fuzziness of p15 enables to regulate PCNA sliding along the DNA that facilitates the switch from replicative to translesion synthesis polymerase binding.

Medical relevance
Overexpression of p15 correlates with tumor progression and poor prognosis in a number of human cancers. The PIP- box/PCNA interaction is a promising target for cancer therapeutics.

Submitted by
Francisco Blanco   fblanco@cicbiogune.es