The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
PCNA binding motifs
Functional site description:
The PCNA binding motifs include the PIP Box, PIP degron and the APIM motif, and are found in proteins involved in DNA replication, repair, methylation and cell cycle control.
ELMs with same func. site: LIG_PCNA_APIM_2  LIG_PCNA_PIPBox_1  LIG_PCNA_yPIPBox_3 
ELM Description:
The PCNA-binding APIM motif was first described in the oxidative demethylase human AlkB homologue 2 (hABH2 or ALKB2) (Gilljam,2009, Bacquin,2013) and later found in the human ZRANB3 protein, which contains an additional PIP Box motif. Both the APIM and PIP Box motifs in ZRANB3 make a significant contribution to PCNA-dependent recruitment of ZRANB3 to DNA-damage sites (Hara,2018). APIM is likely to mediate PCNA binding in several proteins involved in DNA repair and cell cycle control in response to genotoxic stress. The APIM motif bound to PCNA adopts an extended structure with a one-turn 310 helix (Hara,2018, Sebesta,2017). The crystal structures (5YD8 and 5MLW) show that the APIM-binding site on PCNA largely overlaps with the canonical PIP Box binding site (LIG_PCNA_PIPBox_1), forming a helical turn that aligns with the main helix of the PIP Box.
The APIM motif consists of a core helix formed by three hydrophobic residues flanked by positively charged residues and an additional hydrophobic position in the motif N terminus. As in the canonical PCNA-binding PIP Box motif, the core hydrophobic residues of the APIM motif helix dock into a hydrophobic pocket on PCNA. The first of these residues is always aromatic and binds close to a Proline patch in PCNA. So far, no Tryptophan is observed in natural sequences, likely due to its large size. In ZRANB3, the first basic position is engaged in an intramolecular interaction with the backbone carbonyl of Asp1071, while the second basic residue makes a contact with Glu124 in PCNA. The highly conserved hydrophobic position preceding the core of the motif is critical for binding and aligns with the first hydrophobic position of the PIP Box motif, docking into the hydrophobic pocket of PCNA. In the APIM motif, position Ser1070 of ZRANB3 enters the Q-pocket, forming water-mediated hydrogen bonds similar to the Q residue in the PIP Box, but this position is not conserved in the APIM motif and so it is not part of the regular expression.
Pattern: [MLIV].[KR][FY][MLIVF][LIV][KR]
Pattern Probability: 0.0000022
Present in taxon: Metazoa
Interaction Domain:
PCNA_C (PF02747) Proliferating cell nuclear antigen, C-terminal domain (Stochiometry: 1 : 1)
o See 2 Instances for LIG_PCNA_APIM_2
o Abstract
Eukaryotic genome duplication occurs during the DNA synthesis (S) phase of the cell cycle, and ensures the transmission of genetic material to daughter cells. While this process occurs with remarkable fidelity, obstacles such as DNA lesions can lead to replication failure and chromosomes breaks, endangering genome integrity and cell viability [Moldovan,2007]. Several safeguard processes are integrated with DNA replication to sense DNA damage and allow the completion of replication using lower fidelity translesion (TLS) polymerases (Pol ε, ι and κ) or initiate cell apoptosis when the damage can’t be bypassed. DNA synthesis occurs at the replication fork, where PCNA (Proliferating Cell Nuclear Antigen), the "sliding clamp" acts as a scaffolding protein that orchestrates the assembly of replicative DNA polymerases, and integrates DNA damage signalling with DNA repair, by recruiting TLS polymerases to the damage to allow DNA synthesis across DNA lesions [Moldovan,2007].

Many proteins bind to PCNA through PCNA binding motifs, leading to their recruitment to the DNA replication fork. The LIG_PCNA_PIPBox_1 and LIG_PCNA_APIM_2 motifs mediate an interaction with the PCNA PIP Box binding cleft. Among PCNA-binding proteins are enzymes involved in DNA replication, DNA repair and DNA methylation [Choe,2017]. PCNA acts as a scaffold for the integration of DNA replication with cell cycle and DNA damage signalling through the action of cell cycle regulators such as p21, which bind to PCNA using a PIP Box motif [Gulbis,1996]. The PCNA PIP-binding cleft is also a binding site for the related degron motifs DEG_CRL4_CDT2_1 and DEG_CRL4_CDT2_2. The variant PIP degron motif not only interacts with PCNA but also binds the CRL4-Cdt2 ubiquitin ligase through additional interactions, leading to the ubiquitination and proteasomal degradation of PIP degron-containing proteins after DNA damage or during S-phase [Abbas,2008].
The PCNA binding PIP Box and APIM variants target the same binding cleft in PCNA. The classical PCNA binding motif is termed the PIP Box [Warbrick,2000]. The PIP Box motif forms a short 310 helix which interacts with a hydrophobic patch on the outer surface of the clamp through three conserved core hydrophobic positions flanked by an additional residue which is often Q and binds to the conserved “Q pocket” on PCNA (1U7B) [Bruning,2004]. PIP Boxes have a conserved hydrophobic core in vertebrate and fungal proteins, but the fungal motif shows higher sequence variability at some positions, leading to two motif variants. A second variant is the APIM motif [Gilljam,2009], which structurally aligns with the PIP Box helix and has the same conserved hydrophobic interactions but does not bind the Q pocket. Instead, additional interactions of a hydrophobic residue in APIM with human PCNA His44 provide additional binding affinity. While the core hydrophobic positions (⏀xx⏀⏀) are shared between both motifs, additional binding determinants are present which tune the binding affinity and several non-canonical motifs with substitutions at conserved sites exist, highlighting the plasticity of this binding cleft (Prestel,2019).

The range of binding affinities of the PIP Box and APIM motifs covers the low nanomolar (as seen in p21) to the micromolar range and can be strongly modulated by the presence of positive charges in the motif flanking regions, which can increase binding affinity by several orders of magnitude (Prestel,2019). Considering the high number of processes orchestrated by PCNA, the different affinity of each target might function in the fine-tuning of different PCNA functional outputs. Post-translational modifications of PCNA such as ubiquitylation and sumoylation also modulate binding to the PCNA cleft in response to diverse biological stimuli, leading to the recruitment or inhibition of different binding partners (Moldovan,2007, Leung,2018).

The PIP Box is noteworthy in that it is one of the few linear motifs found in all kingdoms of life: The Flap Endonuclease Fen1 (Xni) has a C-terminal PIP Box-like motif in Eubacteria and Archaea as well as in Eukaryotes. Finally, PIP Box and APIM are part of a larger group of PIP-like motifs that includes the translesion synthesis TLS polymerase Rev1-interacting RIR motif (LIG_REV1ctd_RIR_1) and the mismatch repair Mlh1-interacting MIP motif (LIG_MLH1_MIPbox_1) [Ohashi,2009, Gueneau,2013]. These helical motifs have similar consensus sequences that prominently feature two adjacent aromatic residues. PIP-like motifs are functionally interlinked, as they all cooperate in different aspects of DNA repair signalling, and might show an unexpected degree of cross-functionally [Boehm,2016, Boehm,2016].
o 4 selected references:

o 9 GO-Terms:

o 2 Instances for LIG_PCNA_APIM_2
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
1 7 MDRFLVKGAQGGLLRKQEEQ TP 6 Homo sapiens (Human)
1072 1078 RRQSLASKHGSDITRFLVKK TP 7 Homo sapiens (Human)
Please cite: The eukaryotic linear motif resource - 2018 update. (PMID:29136216)

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