LIG_PAM2_2

The PABP-interacting motif 2 (PAM2) directly binds to the MLLE/PABC domain that is found in poly(A)-binding proteins (PABP) like PABPC1 and in members of the HECT domain-containing Hyperplastic Discs (HYD) protein family of E3 ubiquitin ligases (Albrecht,2004, Lim,2006). This domain consists of a conserved bundle of five alpha-helices, of which the N-terminal helix is lacking in some proteins. The interaction with PAM2 sequences involves the most conserved helices 2, 3 and 5, with the exception of yeast, where only helices 2 and 3 mediate binding.
PABPC1 binds to the 3’-poly(A) tail of mRNA molecules via four RNA recognition motifs (RRMs) and recruits different regulatory proteins, which modulate translational activity and mRNA stability, by binding to their PAM2 motifs via its C-terminal MLLE domain (Siddiqui,2007). LIG_PAM2_1 motif-containing PABPC1-binding proteins include Paip1 and Paip2, which stimulate or repress translation by stabilizing or destabilizing, respectively, the closed loop structure of mRNA that is formed by the interaction between PABP and the translation initiation factor eIF4G. Other examples are the eukaryotic release factor eRF3, which contains two overlapping LIG_PAM2_1 motifs, and Tob1 and Pan3, which recruit the deadenylase complexes Caf1-Ccr4 and Pan2-Pan3, respectively, to the mRNA poly(A) tails. These complexes are involved in translation-dependent, eRF3-mediated mRNA decay and translation termination. Tob1 and its family member Tob2 contain two distinct LIG_PAM2_1 motifs, and evidence indicates that in both cases the C-terminal motif is the main interaction site for binding to PABP. PABPC1 binding partners containing the LIG_PAM2_2 motif variant are so far restricted to animal-specific GW182 family proteins, which are part of the RNA-induced silencing complex (RISC) and are essential for microRNA (miRNA)-mediated gene silencing (Jinek,2010). Silencing is achieved by inhibiting translation and promoting degradation of mRNA, and depends on binding of GW182 proteins in the miRNA-loaded RISC to PABPC1. Additional, possibly indirect, interactions also occur between the RRMs of PABPC1 and the C-terminal region of GW182 family members. While in human GW182 proteins PABPC1 binding is predominantly mediated by their PAM2 sequences, in D. melanogaster GW182 the motif, which binds with lower affinity, also contributes to but is dispensable for this interaction in immunoprecipitation experiments (Huntzinger,2010).
The tumor suppressor protein HYD (EDD/UBR5) belongs to the family of HECT domain-containing E3 ubiquitin ligases, which target specific proteins for proteasome-dependent degradation. Evidence suggests that HYD is involved in proliferation and DNA damage signaling. Little is known about the function of the MLLE-PAM2 interaction in HYD E3 ligases, but it has been suggested that PAM2-containing proteins may be targeted for ubiquitination by HYD through binding to its MLLE domain, which is positioned directly adjacent to the catalytic HECT domain. So far, all known PAM2-mediated interactions with HYD involve instances of the LIG_PAM2_1 variant. The structural features of PAM2 binding to the MLLE domain of PABP or HYD are very similar, and similar specificity and binding affinity has been demonstrated for various peptides (Lim,2006).