Sequence-dependent transferability of the LRLLR membrane translocation motif: A computational study of smacN and...

The LRLLR cell-penetrating motif can be transferred to confer membrane translocation activity, but only to compatible recipient peptides. Using umbrella sampling molecular dynamics simulations, we show that C-terminal LRLLR addition to the pro-apoptotic smacN peptide eliminates its translocation barrier, transforming a + 65 kJ/mol barrier into a - 50 kJ/mol energy well. In contrast, N-terminal LRLLR addition to the neuroprotective NR2B9c peptide increases the barrier from +85 to +100 kJ/mol. The LRLLR pentapeptide, identified through systematic screening as essential for spontaneous translocation, represents a minimal penetrating element whose transferability was unknown. We appended this motif to both peptides and calculated potential of mean force profiles across a POPC/POPG bilayer. Analysis of hydrogen bonding patterns, secondary structure, and conformational dynamics reveals the structural basis for these divergent outcomes. Successful transfer to smacN reflects favorable charge complementarity: the hydrophobic, neutral AVPI tetrapeptide provides an ideal platform for the charged, amphipathic LRLLR motif, enabling simultaneous interaction with both membrane leaflets. Transfer failure with NR2B9c stems from its positive charge and conformational rigidity imposed by intramolecular hydrogen bonding, preventing optimal membrane insertion. These findings establish that CPP motif transfer requires compatibility in charge distribution, hydrophobicity, and conformational flexibility, and demonstrate the value of computational screening to identify compatible motif-cargo pairings prior to experimental investment.