Ca²⁺‐dependent interactions between lipids and the tumor‐targeting peptide pHLIP

Abstract

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge‐based design of cancer‐targeting molecular tools, such as pH‐low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond‐timescale all‐atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide‐lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH‐induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+‐induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+‐dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid‐coupled regulation of protein‐membrane insertion by divalent cations.

Arginine Residues Modulate the Membrane Interactions of pHLIP Peptides

“…The first step of the equilibration procedure using these restraints resulted in most peptides’ N-terminus segment converging to the kinked α-helical conformation, similar to what has been shown for the wt peptide . This behavior has also been observed recently using MD simulations and bromolipid quenching experiments (Table S1 of the Supporting Information) . The second step of the protocol consisted of a 100 ns unrestrained constant-pH molecular dynamics (CpHMD) simulation at pH 6.0 to enable residue titration and remove all initial bias, equilibrating both the conformation and protonation states of the titrating residues.…”

“…Several computational studies have focused on simple models, using single lipid membranes, to modulate and understand the physical chemistry of peptide–membrane interactions. These studies delved into modifying the peptide length, charged residues, and hydrophobic stretches to provide molecular insight into a wide range of biophysical phenomena, including peptide structural disposition in the membrane , and the formation of membrane pores. , Although most of these studies do not fully mimic the physiological environment, they provide important information to determine possible folding pathways , and identify key residues for peptide function. − Still, in other experimental studies, complex lipid compositions (i.e., cholesterol and anionic lipids) are often used in tandem with different ion concentrations to highlight how peptide kinetics changes as a result of the more electrostatically charged environments on the protonation changes of the relevant residues. − …”

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

“…[5,[31][32] Numerous examples of utilizing Trp fluorescence to study structure, kinetics and thermodynamics of peptide-lipid interactions exist. [32][33][34][35][36][37][38][39][40][41][42][43][44][45] One of the advantages of using Trp as a probe is that the conformational heterogeneity of the environment can be studies by simultaneously monitoring the position of the emission spectrum and its widths. [31] An illustrative example of the use of the intrinsic Trp fluorescence for examining the protein-membrane interactions is the case study of the membrane-proximal external region (MPER) of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein.…”

Promoting the activity of a receptor tyrosine phosphatase with a novel pH‐responsive transmembrane agonist inhibits cancer‐associated phenotypes

“…Hybrid peptides were prepared by solid-phase peptide synthesis, purified and analyzed by RP-HPLC, and confirmed via MALDI-TOF mass spectrometry, as previously reported (Bloch et al, 2019;Gerhart et al, 2018;Vasquez-Montes et al, 2019;Vasquez-Montes et al, 2022;Wehr et al, 2020).…”