FC0073
Cup  -  eIF4E

Biological function
Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the mRNAs, which are essential for embryonic body axis determination. Cup is involved in repressing at least three localized and developmentally essential mRNAs in Drosophila—oskar, nanos, and gurken—and might function as a general repressor for many as-yet-unknown mRNAs. Cup 4E-BS II has a role in stabilizing the mRNA (by preventing decapping) and simultaneously blocking eIF4G recruitment.

Domain organization/sequence features
Cup is an insect-specific protein of calculated weight 125.7 kDa with no predictable folded domain. In the N-terminal region, Cup contains a canonical eIF4E-binding motif (YXXXXLΦ, where X is any residue and F is hydrophobic). The consensus eIF4E- binding motif is shared by non-homologous eIF4E-binding proteins such as eIF4G, 4E-BPs, 4E-T, and Bicoid. C-terminal to the eIF4E-binding motif of Cup, between the canonical 4E- binding motif and the 4E-T-like region, a second lower- affinity, non- canonical binding region has been mapped by co-precipitation assays. This motif is sufficient to compete with eIF4G for eIF4E binding.

Structural evidence
Cup interacts with eIF4E as two short α-helices connected by a linker that is disordered in the structure. Other portions of the proteins are not present in the structure and are either poorly ordered or were removed during proteolysis. The two stretches, of 35 residues in total, bury 1.2% (350 Ų) of the solvent-accessible surface area of the eIF4E. The N-terminal segment of Cup that is visible in the electron density map includes the eIF4E consensus motif and docks at the convex surface of eIF4E, where it contacts part of the unstructured N terminus of the protein. After a disordered linker of about 20 residues, Cup extends laterally on eIF4E, where a few, mainly hydrophobic, interactions create surface distinct from the 4E-consensus binding site. Both binding sites of Cup are situated away from the m⁷G cap- binding pocket. The binding site I of Cup docks at the convex dorsal surface of eIF4E similarly to the previously described eIF4G and 4E-BPs peptides. Consensus residues (common to 4G and 4E-BP) make similar contacts with several invariant eIF4E side chains found in the helices α1 and α 2 and also with the N-terminal extension of eIF4E. Binding site II of Cup docks laterally with a helix (α 2) that lies antiparallel to the outer β-strand (β2) of eIF4E and a turn that points upward toward the convex surface of eIF4E. Although most of the intermolecular contacts between eIF4E and Cup are conserved, some interactions involve residues that are uniquely conserved in insects. The lateral binding site on eIF4E could be generally exploited by diverse eIF4E-binding proteins.

Biochemical evidence
Two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, non-canonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA.

Mechanism category
Tethering, Flexibility modulation

Significance
Multivalent binding, enabled by the fuzzy linker, contributes significantly to eIF4E stabilization and consequently facilitate preventing the degradation of the mRNA. It is possible that the second, lateral binding site would initiate competition and strengthen the affinity for the mRNA.

Further reading
14685270