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 cyclin dependent kinase (CDK) inhibitor p21 prevents cell cycle progression by blocking the activity of G1 phase CDKs. Upregulation of p21 in response to DNA damage causes cell cycle arrest until DNA is repaired, providing an essential link between genotoxic stress and cell cycle signalling [Bertolin,2015]. The metazoan PCNA binding PIP Box can be represented by the high affinity motif of p21 bound to PCNA (1AXC). The structure reveals two major binding sites formed by the smaller ‘Q pocket’ and a large hydrophobic groove on PCNA [Gulbis,1996]. The ‘Q-pocket’ accommodates the first glutamine residue in LIG_PCNA_PIPBox_1, through van der Waals contacts and two hydrogen bonds to the backbone of Ala252 and Ala208 of PCNA. While Gln provides the highest affinity, the ‘Q pocket’ also accepts hydrophobic residues such as Met in the Ribonuclease H2 subunit B protein RNH2B [2ZVK] [Bubeck,2011] which interacts with Val45, Ala208, Tyr211, and Leu251 of PCNA in a hydrophobic manner.

The core of the motif is formed by hydrophobic and aromatic residues of the ligand (Met147, Phe150 and Tyr151 in p21) also termed the ‘hydrophobic plug’ (⏀xx⏀⏀). These residues adopt an extended conformation with one short 310 amphipathic helix that binds to a hydrophobic pocket on PCNA via van der Waals contacts and proline packing. The first helical turn provides an aliphatic residue, often Leucine. The second helical turn provides two hydrophobic positions that are frequently phenylalanine but almost always aromatic.

Following the core PIP Box helix, there are no required amino acid residues. However, there are usually additional interactions that contribute to the binding affinity, for example beta augmentation backbone interactions [Gulbis,1996]. Many PIP Boxes have positively charged residues after the core helix [Prestel,2019] and, for the PIP Box degrons that also bind the Cdt2 ubiquitin ligase (DEG_CRL4_Cdt2_1 and DEG_CRL4_Cdt2_2), multiple positive charges are always present [Abbas,2008].

Pattern: [QM].[^FHWY][LIVM][^P][^PFWYMLIV](([FYHL][FYW])|([FYH][FYWL]))..
Pattern Probability: 0.0000866
Present in taxons: cellular organisms Eukaryota
Interaction Domain:
PCNA_C (PF02747) Proliferating cell nuclear antigen, C-terminal domain (Stochiometry: 1 : 1)
PDB Structure: 1U76
o See 19 Instances for LIG_PCNA_PIPBox_1
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 14 selected references:

o 8 GO-Terms:

o 19 Instances for LIG_PCNA_PIPBox_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P52701 MSH6
4 13 MSRQSTLYSFFPKSPALSDA TP 5 Homo sapiens (Human)
P18858 LIG1
2 11 MQRSIMSFFHPKKEGKAKKP TP 7 Homo sapiens (Human)
P39748 FEN1
337 346 RQGSTQGRLDDFFKVTGSLS TP 6 Homo sapiens (Human)
P26358 DNMT1
164 173 RKSTRQTTITSHFAKGPAKR TP 10 Homo sapiens (Human)
Q15054 POLD3
456 465 TAALGKANRQVSITGFFQRK TP 2 Homo sapiens (Human)
O94762 RECQL5
964 973 VKEEAQNLIRHFFHGRARCE TP 1 Homo sapiens (Human)
P20585 MSH3
21 30 SAPARQAVLSRFFQSTGSLK TP 3 Homo sapiens (Human)
701 710 KRPRPEGMQTLESFFKPLTH TP 5 Homo sapiens (Human)
523 532 KPRMGQQVLDNFFRSHISTD TP 4 Homo sapiens (Human)
788 797 NKPGLQIKLNELWKNFGFKK TP 3 Homo sapiens (Human)
390 399 GSSRGQKNLKSYFQPSPSCP TP 3 Homo sapiens (Human)
460 469 VRVKTVPSLFQAKLDTFLWS TP 9 Homo sapiens (Human)
519 528 FEKEKQHDIRSFFVPQPKKR TP 1 Homo sapiens (Human)
294 303 VDKSGMKSIDTFFGVKNKKK TP 5 Homo sapiens (Human)
Q15004 PCLAF
62 71 PTPKWQKGIGEFFRLSPKDS TP 7 Homo sapiens (Human)
325 334 VSNSHQNVLSNYFPRVSFAN TP 3 Homo sapiens (Human)
P28715 ERCC5
990 999 DAQQTQLRIDSFFRLAQQEK TP 1 Homo sapiens (Human)
P38936 CDKN1A
144 153 GRKRRQTSMTDFYHSKRRLI TP 1 Homo sapiens (Human)
P13051 UNG
4 13 MIGQKTLYSFFSPSPARKRH TP 1 Homo sapiens (Human)
Please cite: The eukaryotic linear motif resource - 2018 update. (PMID:29136216)

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