Deprotonation of C-terminal Acidic Residues Holds the Key to the Exit Pathway of pHLIP

Abstract

pH Low Insertion Peptide (pHLIP) is acid-sensitive membrane active peptide that inserts into the membrane under acidic conditions. This functionality makes pHLIP a promising candidate for early-stage diagnostic imaging of and targeted drug delivery to disorders associated with acidosis. pHLIP is known to exist in three states: coiled in solution (state I), bound to the membrane (state II), both at pH≥7, and inserted as a transmembrane ɑ-helix (state III) and pH≤ 5.3. The transition from state II to state III is triggered by protonation of the aspartic and glutamic acid residues in the presence of a membrane environment. Although state III has been extensively characterized via experimental and computational techniques [1,2], the membrane-peptide interactions initiating the exit of pHLIP are poorly understood. We hypothesize that deprotonation of C-teminal acidic residues is the critical controlling factor towards the exit of pHLIP. To test this hypothesis, we used molecular dynamics (MD) simulations to model pHLIP inserted in a 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) bilayer, using the protonation states as our variable. We find that fully protonated pHLIP remains stable, however, deprotonation of acidic residues induces a loss of helical conformation. Upon deprotonation of the C-terminal acidic residues (D31, D33, and E34), extensive snorkeling of waters into the interior of the membrane and a decrease in the ordering of the lipid tails is observed, indicating the destabilization of the membrane-peptide interface. Together, these are the hallmarks of the initial stages in the exit of pHLIP from the membrane, which contribute to our fundamental understanding of pHLIP function which will ultimately lead to optimized variants for therapeutic and diagnosis applications. [1] Otieno et. al PNAS 2018. 115 (48) 12194-12199. [2] Machuqueiro et. al JCTC 2018. 14(6):3289-3297.