Sequence-dependent transferability of the LRLLR membrane translocation motif: A computational study of smacN and NR2B9c peptides.

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 demonstrate that C-terminal LRLLR addition to the pro-apoptotic smacN peptide eliminates its translocation barrier entirely, 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 translocation barrier from +85 to +100 kJ/mol, demonstrating that motif transfer can prove counterproductive for incompatible sequences. Cell-penetrating peptides offer promising strategies for intracellular delivery of therapeutic cargo, yet the sequence determinants governing their activity remain incompletely understood. The LRLLR motif, identified through systematic screening as essential for spontaneous membrane translocation, represents a minimal penetrating element whose transferability has not been previously evaluated. We appended this motif to two clinically relevant peptides: smacN, a tetrapeptide targeting inhibitor of apoptosis proteins in chemotherapy-resistant cancers, and NR2B9c, a nonapeptide that disrupts excitotoxic signaling in ischemic stroke. Potential of mean force profiles calculated across a POPC/POPG bilayer, combined with analysis of hydrogen bonding patterns, secondary structure propensity, and conformational dynamics, reveal the structural basis for these divergent outcomes. Successful transfer to smacN results from favorable complementarity: the hydrophobic, neutral smacN provides an ideal platform for the charged, amphipathic LRLLR motif, yielding a chimera capable of simultaneous interaction with both membrane leaflets. Transfer failure with NR2B9c stems from conformational rigidity induced by intramolecular hydrogen bonding, which prevents optimal membrane insertion, combined with unfavorable positioning of internal polar residues at the bilayer center. These findings establish that cell-penetrating motif transfer requires compatibility in charge distribution, hydrophobicity, and conformational flexibility between the motif and recipient sequence. The smacN-LRLLR chimera emerges as a promising candidate for experimental validation as a membrane-permeable therapeutic for survivin-positive tumors. More broadly, this work demonstrates the value of computational screening to identify compatible motif-cargo pairings prior to experimental investment.