Elucidating the Connection between Protonation Switches and Binding and Folding of pHLIP

Abstract

pH (Low) Insertion Peptide (pHLIP) is a soluble peptide that binds and inserts into cell membranes in a pH-sensitive manner. pHLIP is unstructured at alkaline to neutral pH and binds to the surface of membrane bilayers. At acidic pH, it simultaneously folds into an α-helix and inserts into the bilayer through its C-terminus. Although extensively studied, pHLIP-membrane interactions are not well understood at the molecular level, which severely impairs the development of pHLIP as a potential delivery mechanism for cancer chemotherapeutics. We have employed molecular dynamics (MD) simulations to investigate the behavior of pHLIP in solution and during the binding process to the lipid bilayer surface. Using a total of 820 ns trajectory, we have generated a pool of conformations of unstructured pHLIP, and clustered them using k-means clustering. In addition, our subsequent simulations of these pHLIP structures bound to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid surface agree well with recently published solid-state NMR results [1]. The N-terminus of pHLIP preferentially binds to the POPC surface, initiated by a combination of partitioning of hydrophobic stretches of the peptide beneath the headgroup region and hydrogen-bonding of the polar R11 sidechain with the PC headgroups. It has also been determined that D14 and D25 play a significant role in governing the pH response of pHLIP [2]. We have discovered that the altered apparent pK of insertion and sharpness of transition of unnatural amino acid mutations of pHLIP at these positions may be linked to changes in their helix-forming propensity in solution. These results are key to our understanding of pHLIP-membrane interactions and provide a first step in an atomistic characterization of the binding, folding, and insertion processes of pHLIP. [1] Shu, Nat. Comm., 6:7787 (2015); [2] Onyango, Ange. Chem., 54:3658 (2015).