(A) Sequence of MLLE-binding PAM2 motifs. PAM2-N and PAM2-C of eRF3 are aligned against sequences from Tob (transducer of Erb1), poly(A) specific ribonuclease 3 (PAN3), PABP-interacting protein 2 (Paip2) and Ataxin-2. Residues in the overlap are underlined. The consensus of most conserved residues that contribute to the MLLE/PAM2 binding is shown: Φ represents a hydrophobic residue [12]. (B) Overlaid structures of the complexes of eRF3 PAM2-N (yellow) and PAM2-C (magenta) bound to the MLLE domain (grey) from PABPC1. eRF3 Phe76 shifts position and binds to either MLLE helix α2/α3 in the PAM2-N complex or helix α3/α5 in the PAM2-C complex. Leu69 or Phe85 then occupies the vacated hydrophobic binding site. (C) Stick representation of overlaid structures of eRF3 PAM2-N (yellow) and PAM2-C (magenta) bound to the MLLE domain from PABPC1 (green).

The mRNA poly(A) binds multiple PABPC1 proteins through the N-terminal RRM1-4 domains. The C-terminal MLLE domain of PABPC1 (open circle missing a triangle) binds proteins containing PAM2 motifs (open triangles). The higher affinity PABPC1-eRF3 interaction inhibits the recruitment of two mRNA deadenylation complexes, PAN2/3 and Ccr4-Not-Caf1, to the mRNA. These complexes bind the MLLE binding via the PAM2 motifs of PAN3 and Tob, a member of the antiproliferative BTG family. The MLLE-PAM2 interaction is required for PABPC1-dependent stimulation of deadenylase activity by both PAN3 and Tob [26], [31].

(A) Cartoon representation of the MLLE domain (green) with eRF3 PAM2-N (yellow). (B) Details of the eRF3 PAM2-N structure. The eRF3 peptide makes a β-turn stabilized by hydrogen bonds between carbonyl of Asn70 and amide of Ala73 and between side chain of Asn70 and amide of Asn72. Mlle binding is mediated by several hydrophobic interactions: Leu69 binding to the pocket formed by Leu585, Ala610, and the aliphatic portion of Lys606 of MLLE, Val71 inserting into a smaller pocket formed by the side chains of Met584, Val613, Leu614 and His617, and Ala73 contacting the Met584 side chain. Intermolecular hydrogen bonds occur between the side chain of MLLE Lys580 with carbonyls of Val71 and Ala73 of Paip2. (C) C-terminal portion of PAM2-N. Phe76 binds the shallow pocket formed by Thr582, Phe567, Leu586 and Gly563 of MLLE. The amide of this phenylalanine is engaged in hydrogen bonding with carbonyl of MLLE Gly579. Pro78 of eRF3 interacts with the aromatic ring of Phe567. Side chains of Gln560 and Glu564 of MLLE form a network of intermolecular hydrogen bonds with the amides of Val77 and Asn79 of eRF3. (D) Cartoon representation of the MLLE domain (green) with eRF3 PAM2-C (magenta). (E) Details of the eRF3 PAM2-C structure. The aromatic side chain of eRF3 Phe76 binds in the hydrophobic cavity previously occupied by Leu69 of the PAM2-N peptide, while Pro78 interacts with the side chain of Val617. Side chain of Asn79 also makes a salt bridge with MLLE Glu587. (F) C-terminal portion of PAM2-C. The interactions of eRF3 Phe85 and Pro87 with MLLE are identical to those of Phe76 and Pro78 in the PAM2-N structure. The side chain of Glu84 of eRF3 forms intermolecular hydrogen bonds with amides of Gly579 and Lys580 via an ordered water molecule. Hydrogen bonds are shown in black, ordered water molecules in cyan. Figures were made with PyMOL (http://pymol.sourceforge.net/).