pH-triggered membrane permeabilizing peptides could be exploited in a variety of applications, such as to enable cargo release from endosomes for cellular delivery, or as cancer therapeutics that selectively permeabilize the plasma membranes of malignant cells. Such peptides would be especially useful if they could enable the movement of macromolecules across membranes, a rare property in membrane permeabilizing peptides. Here we approach this goal by using an orthogonal high-throughput screen of an iterative peptide library to identify peptide sequences that have the following two properties: (i) little synthetic lipid membrane permeabilization at physiological pH 7 at high peptide concentration and (ii) efficient formation of macromolecule-sized defects in synthetic lipid membranes at acidic pH 5 and low peptide concentration. The peptides we selected are remarkably potent macromolecular sized pore-formers at pH 5, while having little or no activity at pH 7, as intended. The action of these peptides likely relies on tight coupling between membrane partitioning, α-helix formation, and electrostatic repulsions between acidic sidechains, which collectively drive a sharp pH-triggered transition between inactive and active configurations with apparent pKa values of 5.5–5.8. This work opens new doors to developing applications of peptides with membrane permeabilizing activities that are triggered by physiologically relevant decreases in pH.

Edited by Wolfgang Peti The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.

Using synthetic molecular evolution, we previously discovered a family of peptides that cause macromolecular poration in synthetic membranes at low peptide concentration in a way that is triggered by acidic pH. To understand the mechanism of action of these “pHD peptides”, here we systematically explored structure–function relationships through measurements of the effect of pH and peptide concentration on membrane binding, peptide structure, and the formation of macromolecular-sized pores in membranes. Both AFM and functional assays demonstrate the peptide-induced appearance of large pores in bilayers. Pore formation has a very steep pH dependence and is also dependent on peptide concentration. In vesicles, 50% leakage of 40 kDa dextrans occurs at 1 bound peptide per 1300 lipids or only 75 peptides per vesicle, an observation that holds true across a wide range of acidic pH values. The major role of pH is to regulate the amount of peptide bound per vesicle. The physical chemistry and sequence of the pHD peptides affect their potency and pH dependence; therefore, the sequence–structure–function relationships described here can be used for the future design and optimization of membrane permeabilizing peptides for specific applications.

No abstract

No abstract