Elucidating the Lipid Binding Properties of Membrane-Active Peptides Using Cyclised Nanodiscs

Abstract

The lipid composition of the cellular membrane plays an important role in a number of biological processes including the binding of membrane-active peptides. Characterization of membrane binding remains challenging, due to the technical limitations associated with the use of standard biophysical techniques and available membrane models. Here, we investigate the lipid binding properties of two membrane-active peptides, VSTx1, a well characterized ion-channel inhibitor, identified from spider venom, that preferentially binds to anionic lipid mixtures, and AA139 an antimicrobial β-hairpin peptide with uncharacterised lipid binding properties, currently in pre-clinical development. The lipid binding properties of these peptides are elucidated using nanodiscs formed by both linear and circularized (sortase-mediated) forms of a membrane scaffold protein (MSP1D1ΔH5). We find that nanodiscs formed by circularized MSPs—in contrast to those formed by linear MSPs—are sufficiently stable under sample conditions typically used for biophysical measurements (including lipid composition, a range of buffers, temperatures and concentrations). Using these circularized nanodiscs, we are able to extract detailed thermodynamic data using isothermal titration calorimetry (ITC) as well as atomic resolution mapping of the lipid binding interfaces of our isotope labeled peptides using solution-state, heteronuclear, nuclear magnetic resonance (NMR) spectroscopy. This represents a novel and general approach for elucidating the thermodynamics and molecular interface of membrane-active peptides toward flat lipid bilayers of variable composition. Our approach is validated by first determining the thermodynamic parameters and binding interface of VSTx1 toward the lipid bilayer, which shows good agreement with previous studies using lipid micelles and liposomes. The method is then applied to AA139, where the membrane binding properties are unknown. This characterization, involved solving the high-resolution structure of AA139 in solution using NMR spectroscopy and the development of a suitable expression system for isotope labeling. AA139 was found to bind exclusively to anionic membranes with moderate affinity ( K d ~low μM), and was found to have a lipid binding interface involving the termini of the β-hairpin structure. The preference of AA139 for anionic lipids supports a role for membrane binding in the mode-of-action of this peptide, which is also consistent with its higher inhibitory activity against bacterial cells compared to mammalian cells. The described approach is a powerful method for investigation of the membrane binding properties of this important class of molecules.

Structural Modeling of Peptide Toxin - Ion Channel Interactions using RosettaDock

“…Experimental data highlights the importance of membrane embedding in the function of some of the peptide toxins tested in our study, particularly for GMTs (Agwa et al, 2017;Henriques et al, 2016;Lee and MacKinnon, 2004;Mihailescu et al, 2014;Mueller et al, 2020;Zhang et al, 2019). We took advantage of Rosetta's versatility and implemented the latest membrane energy function (franklin2019) in our modeling protocol to evaluate its performance compared to the standard energy function (REF2015).…”

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

“…The production of monomeric cNDs with a 9 nm diameter (cNW9-derived from MSP1D1∆H4H5 or MSP9) has proven to be particularly challenging 9,31 , we therefore chose this as a test system to compare the effect of the different linkers on the efficiency of the autocyclase system.…”

“…Head-to-tail macrocyclisation is a naturally occurring post-translational modification that stabilises the protein fold, leading to enhanced thermal stability and resistance to proteolytic digestion by exoproteases [1][2][3][4] . Mimicry of this natural phenomenon has and continues to inspire protein engineering efforts 5,6 including macrocyclic peptide drug leads 7 and highly stable cyclised lipid nanodiscs used in biophysical characterisation of membrane proteins and membrane active peptides 8,9 .…”

Small molecules enhance the potency of natural antimicrobial peptides

“…Notably, all bacterial membranes have anionic lipids in their composition which can be PG and/or CL. The presence of these anionic lipids explains the selective toxicity of AMPs against bacteria but not against mammalian cells [18], Simple phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) models representing neutral mammalian membranes and negatively charged bacterial membranes are routinely used in studies to easily represent differences between the two systems (see for example [19][20][21] and our previous work on AMPs [22]. Therefore, we chose to use POPC and POPG, consistent with a recent simulation study on LL-37 [23].…”

“…Simple phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) models representing neutral mammalian membranes and negatively charged bacterial membranes are routinely used in studies to easily represent differences between the two systems (see for example [19–21] and our previous work on AMPs [22]. Therefore, we chose to use POPC and POPG, consistent with a recent simulation study on LL-37 [23].…”