Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the <i>E. coli</i> rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

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Publication Details

Journal
eLife
Published
2025-01-16
DOI
https://doi.org/10.7554/elife.104237
Primary Topic
ATP Synthase and ATPases Research
Type
article
Field-Weighted Citation Impact
0.00

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article

Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization

Carol V. Robinson, Erik Lindahl, Rebecca J. Howard, Robin Adam Corey et al.
eLife
ATP Synthase and ATPases Research
article

Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization

Carol V. Robinson, Erik Lindahl, Rebecca J. Howard, Robin Adam Corey, Kvido Střı́šovský, Abraham Olusegun Oluwole, David Drew, Mia Abramsson, Jan Škerle, Olivia Andén, Erik G. Marklund, Phillip J. Stansfeld, Louise J Persson, Michael Landreh
article en

Abstract

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the <i>E. coli</i> rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

eLifeVol. 14
Uppsala University (SE), Stockholm University (SE), Science for Life Laboratory (SE), Karolinska Institutet (SE), University of Bristol (GB), University of Warwick (GB), University of Oxford (GB), Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry (CZ), KTH Royal Institute of Technology (SE)
Eli Lilly and Company, Swedish e-Science Research Centre, Ministerstvo Školství, Mládeže a Tělovýchovy, Cancerfonden, Svenska Sällskapet för Medicinsk Forskning, Karolinska Institutet, Kungliga Tekniska Högskolan, Vetenskapsrådet, Medical Research Council, Engineering and Physical Sciences Research Council, European Regional Development Fund
Openalex Percentile: Top 99%
ATP Synthase and ATPases Research
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