Progress in peptide scanning techniques opens new perspectives for peptide drug development. It comes with the promise of a paradigm change in peptide drug design in which peptide drugs will be closer to the parent peptides. However, scanning still remains assimilable to a trial and error strategy that could benefit from being combined with specific in silico approaches that start reaching maturity.

Background:We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions.Method:Here, we have studied the interactions between recombinant eL42 and eRF1 proteins and the tRNA substrate by means of the Biacore assay, using the wild-type eL42 protein, the eL42-Δ(GGQTK) mutant (the eL42 protein whose GGQTK motif has been deleted), the single Q51E and K53Q mutants (eL42-Q51E and eL42-K53Q, respectively), as well as the double Q51A/K53A mutant (eL42-Q51A/K53A).Results:Our results show that the monomethylated Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence of eL42 and the monomethylated GGQ motif of eRF1 represents the sites of interaction between these two proteins through hydrophobic contacts between methyl groups. We also demonstrate that the interactions between eL42 and tRNA or 28S rRNA are characterized by strong binding affinities (KD values in the nanomolar or picomolar range, respectively) which argue for specific interactions. Strong interactions between eL42 and tRNA are likely to be responsible for the decrease in the poly(U)-dependent poly(Phe) synthesis activity of human 80S or E. coli 70S ribosomes in the presence of added human recombinant eL42. It is proposed that the decrease of the activity of the ribosome is caused by the sequestration of the substrate Phe-tRNAPhe by the added eL42 protein.Conclusion:Interactions between the monomethylated Gln-51 and Lys-53 residues of the 49GGQTK53 motif of the human eL42 protein and the methylated GGQ motif of eRF1 are likely to play a functional role on translating human 80S ribosomes.

The blind identification of candidate patches of interaction on the protein surface is a difficult task that can hardly be accomplished without a heuristic or the use of simplified representations to speed up the search. The PEP-SiteFinder protocol performs a systematic blind search on the protein surface using a rigid docking procedure applied to a limited set of peptide suboptimal conformations expected to approximate satisfactorily the conformation of the peptide in interaction. All steps rely on a coarse-grained representation of the protein and the peptide. While simple, such a protocol can help to infer useful information, assuming a critical analysis of the results. Moreover, such a protocol can be extended to a semi-flexible protocol where the suboptimal conformations are directly folded in the vicinity of the receptor.