The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
MLH1 C-terminal Domain Ligand
Functional site description:
A number of different DNA replication and repair pathways co-operate to ensure the reliable repair of different DNA damage types. MutS and MutL are central players of the mismatch repair pathway. MutLα is a heterodimer composed of Mlh1 and Pms1 in yeast and of MLH1 and PMS2 in humans. Mlh1 acts as a major signal integrator; its C-terminal domain has two non-overlapping sites for the binding of partners. The site of Mlh1 critical for the Mlh1/Pms1 heterodimerization and interactions essential in mismatch repair is different from the one responsible for the interaction with partners not essential for mismatch repair, for example the Exo1 (Exodeoxyribonuclease 1), Ntg2 (DNA N-glycosylase and apurinic or apyrimidinic lyase), and Sgs1 (DNA helicase of the RecQ family) proteins in yeast and EXO1 and BLM in human. The latter partners interact with the Mlh1 CTD through a widely conserved MLH1 interacting peptide motif, the MIP-box.

ELM Description:
In the two available Mlh1-Pms1-MIP-box ternary complexes (4FMN and 4FMO), the Ntg2 and Exo1 MIP-box fragments adopt an ST-turn conformation with the side chain of the MIP-box serine forming an intramolecular hydrogen bond with the main chain NH group of the residue in position (i+2) in the peptide (Gueneau,2013). The serine residue also contributes with important interface interactions. It makes a hydrogen bond with the hydroxyl moiety of Mlh1 Tyr630 as well as with the side chain of Glu682 (Gueneau,2013). The two aromatic residues of the MIP-box insert into hydrophobic pockets. The first aromatic, (Tyr26 in Ntg2 or Phe447 in Exo1), is in contact with Mlh1 residues Met626 and Tyr630. Additionally, the hydroxyl group of Tyr26 in Ntg2 makes a hydrogen bond with the Mlh1 Glu629 side chain. The second aromatic residue in the MIP-box motif is a phenylalanine (Phe27 for Ntg2 and Phe449 for Exo1), which is fully buried in a cavity surrounded by residues Met626, Tyr630, Cys685 and Leu686 of Mlh1 (Gueneau,2013). Accordingly, mutational studies have confirmed that the serine and the two aromatic residues in the MIP-box are crucial for the interaction (Gellon,2002, Dherin,2009).
With three well-defined positions, the MIP-box is a relatively confident motif. Furthermore, in the positions preceding and following the serine positively charged residues were observed in most instances of the motif. Among the MIP-box containing Mlh1 partners, Exo1 is universally conserved among eukaryotes. The Exo1 MIP-box sequence alignment shows that the serine and the two aromatic residues are absolutely conserved, while the positions preceding and following the serine are somewhat floppy. There is a preference for positively charged residues (which is stronger in the position following the serine), but other residues also occur (Dherin,2009). Accordingly, mutating these residues to Ala in Ntg2 somewhat weakened but did not abolish the interaction (Gellon,2002).
Pattern: .S.[FY][F]
Pattern Probability: 0.0000621
Present in taxon: Eukaryota
Interaction Domain:
Mlh1_C (PF16413) DNA mismatch repair protein Mlh1 C-terminus (Stochiometry: 1 : 1)
o See 6 Instances for LIG_MLH1_MIPbox_1
o Abstract
Organisms have developed robust DNA repair mechanisms to prevent the deleterious consequences of different types of DNA damage. The base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways cooperate in the repair of oxidative DNA damage through protein-protein interactions (Gellon,2002), while the MMR pathway is also responsible for correcting base-base mismatches and insertion/deletion loops arising during DNA replication (Dherin,2009). MutL and MutS are essential factors of MMR and meiotic recombination. MutLα is a heterodimer composed of Mlh1 and Pms1 in yeast and of MLH1 and PMS2 in humans. This dimer is believed to act as a scaffold that links the proteins that recognize the mismatches in the DNA with the proteins that carry out the subsequent steps of MMR. Mlh1 interacts through its C-terminal domain (CTD; PF16413, Mlh1_C Domain) with MutL homologs and proteins involved in DNA repair and replication. One can distinguish two different groups among the partners of the Mlh1 CTD, which employ two different, non-overlapping binding sites for Mlh1 interaction: Pms1, Mlh2, and Mlh3, which are required for specialized MMR functions, interact with site 1 while Exo1, Sgs1, and Ntg2, which are not strictly required for MMR interact with site 2 (Gellon,2002). The MIP-box is a widely conserved short sequence motif that is specific for the partners binding site 2. The motif is composed of a conserved serine residue followed by a preferentially positively charged residue and two aromatic residues, with the second being a phenylalanine. The interactions of MIP-box motifs in different replication and repair proteins with Mlh1 CTD have been confirmed by diverse methods, including two-hybrid studies, pull-down assays, alanine-scanning (Gellon,2002), ITC measurements (Dherin,2009) and the structures of two complexes are also available (Gueneau,2013). X-ray crystal structures of Mlh1 bound to MIP motifs from Exo1 (4FMO) and Ntg2 (4FMN) show that the MIP peptides adopt an ST turn conformation in which the side chain of the serine forms a hydrogen bond with the main chain amine group of the first aromatic residue (Gueneau,2013). Residues in the MIP motif important for Mlh1 binding include the serine and the two aromatic residues. These latter residues bind in hydrophobic pockets (Gellon,2002, Dherin,2009, Gueneau,2013).
Due to the presence of two different sites, Mlh1 can form heterotrimers by binding a MutL homolog and an S2-interacting protein at the same time, for example, the Mlh1-Pms1-Exo1 heterotrimer (Tran,2001). Binding multiple partners at sites S1 and S2 may result in the formation of a variety of binary and ternary complexes, whose biological impact must be diverse. Therefore Mlh1 acts as a “molecular matchmaker,” a major coordinator, and regulator of the MMR proteins and coupling this pathway to replication machinery (Gellon,2002).
In the large network of interacting proteins responsible for genome maintenance, PCNA, Rev1, Mlh1, and pol δ are hubs responsible for the perception and integration of different signals and for the orchestration of adequate responses. Many proteins interact with these hubs via short sequence motifs such as PIP box motifs, RIR motifs, MIP-box motifs, and F1 motifs, respectively. The traditional paradigm is that these motifs are distinct entities, each specific for binding a single target protein. However, the motifs have consensus sequences that involve two adjacent aromatic residues that facilitate the interactions with target proteins by binding within hydrophobic pockets. Interestingly, several studies have challenged this paradigm by showing that some of these simple double-aromatic residue motifs are specific for more than one target protein (Boehm,2016). For example, the RIR motif of human polymerase η interacts with both Rev1 and pol δ (Baldeck,2015). Similarly, the PIP motif of yeast pol η interacts with both PCNA and Rev1 (Boehm,2016). Furthermore, the PCNA-binding motif on the N-terminus of yeast MutSβ subunit Msh3 was demonstrated to be critical for MutSβ – MutLα binding and thus it presumably also acts as a MIP motif binding to the Mlh1 subunit of MutLα (Mlh1-Pms1 heterodimer) (Iyer,2010). The ability of these peptide motifs to bind more than one target proteins may necessitate to re-think their strict classifications and to introduce a broader class of PIP-like motifs (Boehm,2016), perhaps with molecular switching properties.
o 6 selected references:

o 5 GO-Terms:

o 6 Instances for LIG_MLH1_MIPbox_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P25336 MSH3
7 11 MAGQPTISRFFKKAVKSELT TP 5 Saccharomyces cerevisiae (Baker"s yeast)
P39875 EXO1
444 448 QETLKDTRSKFFNKPSMTVV TP 6 Saccharomyces cerevisiae S288c
Q08214 NTG2
23 27 DIEEVEVRSKYFKKNERTVE TP 7 Saccharomyces cerevisiae S288c
P54132 BLM
1322 1326 LDEEIPVSSHYFASKTRNER TP 2 Homo sapiens (Human)
503 507 AVVVPGTRSRFFCSSDSTDC TP 3 Homo sapiens (Human)
P35187 SGS1
1382 1386 IAQSTGTKSKFFGANLNEAK TP 3 Saccharomyces cerevisiae S288c
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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