Abstract

The PABP-interacting Motif PAM2 has a length of 13 amino acids and directly binds to the MLLE (previously known as PABC) peptide-binding domain that is found in poly(A)-binding proteins (PABP) and in members of the HECT domain-containing Hyperplastic Discs (HYD) protein family of E3 ubiquitin ligases. This domain consists of a conserved bundle of five alpha-helices, of which the N-terminal helix is lacking in some proteins.
PABP binds to the 3'-poly(A) tail of mRNA molecules via 4 RNA recognition motifs at its N terminus, and recruits different proteins that modulate mRNA stability and translational activity via its C-terminal MLLE domain that binds to the PAM2 motif in these proteins. These 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 PAM2 motifs, and Tob1 and Pan3, which recruit the deadenylase complexes Caf1-Ccr4 and Pan2-Pan3, respectively, to the 3'-poly(A) tail of mRNA's. These complexes are involved in translation-dependent, eRF3-mediated mRNA decay and translation termination. Tob1 and its family member Tob2 contain 2 distinct PAM2 motifs, and evidence indicates that in both cases the C-terminal PAM2 motif is the main interaction site for binding to PABP.
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. 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.
The MLLE domain consists of a conserved bundle of five alpha-helices. However, some MLLE domain-containing proteins lack the N-terminal helix.
The PAM2 sequence binds to the most conserved helices 2, 3 and 5 of the MLLE domain, with the exception of the distinct yeast PAM2 motif, which only interacts with helices 2 and 3. Binding of PAM2 to the MLLE domain mainly involves hydrophobic interactions, the most important of which are mediated by the residue at position 3, most frequently a leucine, and, primarily, by the residue at position 10, most frequently a phenylalanine. These residues occupy 2 hydrophobic pockets on the MLLE domain. Another important interaction is mediated by the invariant alanine at position 7 of the PAM2 motif.