Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition

TY - JOUR

T1 - Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition

AU - Chen, Charles H.

AU - Starr, Charles G.

AU - Guha, Shantanu

AU - Wimley, William C.

AU - Ulmschneider, Martin B.

AU - Ulmschneider, Jakob P.

N1 - Funding Information: We thank the Karlsruhe Institute of Technology (KIT) ANKA synchrotron CD beamline staff for support and beamtime. We thank Jochen Bürck at KIT for valuable discussion and technical support for ANKA synchrotron CD beamline. We thank Guangshun Wang at the University of Nebraska Medical Center for providing the raw data of the antimicrobial peptide database. We thank Katherine Tripp at the Center for Molecular Biophysics, Johns Hopkins University for helping the experimental setup for isothermal titration calorimeter. We thank Jodie Franklin at the Synthesis and Sequencing Facility at the Johns Hopkins University School of Medicine for sequencing the LDKA peptides. We thank Kalina Hristova and Honggang Cui at Johns Hopkins University and Gregory Wiedman at Seton Hall University and Bonnie Wallace at Birkbeck, University of London and Matthew Upton at University of Plymouth and Alexey Ladokhin at University of Kansas for valuable discussions. Simulation resources were supported by the MARCC supercomputer facility at Johns Hopkins University. Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/2

Y1 - 2021/2

N2 - The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure–function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization. Graphic Abstract: [Figure not available: see fulltext.]

AB - The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure–function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization. Graphic Abstract: [Figure not available: see fulltext.]

KW - Antimicrobial peptides

KW - Bacterial selectivity

KW - Drug-resistant bacteria

KW - Leucine-rich peptide

KW - Pore formation

KW - Protein folding

UR - http://www.scopus.com/inward/record.url?scp=85100766541&partnerID=8YFLogxK

U2 - 10.1007/s00232-021-00174-1

DO - 10.1007/s00232-021-00174-1

M3 - Article

AN - SCOPUS:85100766541

SN - 0022-2631

VL - 254

SP - 75

EP - 96

JO - Journal of Membrane Biology

JF - Journal of Membrane Biology

IS - 1

ER -