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 E. coli 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.

Department for Cell and Molecular Biology Uppsala University Uppsala Sweden

Department of Applied Physics Science for Life Laboratory KTH Royal Institute of Technology Solna Sweden

Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Solna Sweden

Department of Biochemistry and Biophysics Stockholm University Stockholm Sweden

Department of Chemistry BMC Uppsala University Uppsala Sweden

Department of Chemistry University of Oxford Oxford United Kingdom

Department of Microbiology Tumor and Cell Biology Karolinska Institutet Solna Sweden

Institute of Organic Chemistry and Biochemistry Academy of Science of the Czech Republic Prague Czech Republic

Kavli Institute for Nanoscience Discovery University of Oxford Oxford United Kingdom

School of Life Sciences and Chemistry University of Warwick Coventry United Kingdom

School of Physiology Pharmacology and Neuroscience University of Bristol Bristol United Kingdom

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doi: 10.1101/2024.05.27.592301 PubMed

Před aktualizací

doi: 10.7554/eLife.104237.1 PubMed

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doi: 10.7554/eLife.104237.2 PubMed

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Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. doi: 10.1016/j.softx.2015.06.001. DOI

Abraham M, Alekseenko A, Basov V, Bergh C, Briand E, Brown A, Doijade M, Fiorin G, Fleischmann S, Gorelov S, Gouaillardet G, Grey A, Irrgang ME, Jalalypour F, Jordan J, Kutzner C, Lemkul JA, Merz P, Miletic V, Morozov D, Nabet J, Pall S, Pasquadibisceglie A, Pellegrino M, Santuz H, Schulz R, Shugaeva T, Shvetsov A, Villa A, Wingbermuehle S, Hess B, Lindahl E. GROMACS 2024-beta manual. 1.0Zenodo. 2023 doi: 10.5281/zenodo.3957257. DOI

Akdel M, Pires DEV, Pardo EP, Jänes J, Zalevsky AO, Mészáros B, Bryant P, Good LL, Laskowski RA, Pozzati G, Shenoy A, Zhu W, Kundrotas P, Serra VR, Rodrigues CHM, Dunham AS, Burke D, Borkakoti N, Velankar S, Frost A, Basquin J, Lindorff-Larsen K, Bateman A, Kajava AV, Valencia A, Ovchinnikov S, Durairaj J, Ascher DB, Thornton JM, Davey NE, Stein A, Elofsson A, Croll TI, Beltrao P. A structural biology community assessment of AlphaFold2 applications. Nature Structural & Molecular Biology. 2022;29:1056–1067. doi: 10.1038/s41594-022-00849-w. PubMed DOI PMC

Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics. 1984;81:3684–3690. doi: 10.1063/1.448118. DOI

Best RB, Zhu X, Shim J, Lopes PEM, Mittal J, Feig M, MacKerell AD., Jr Optimization of the additive CHARMM All-Atom protein force field targeting improved sampling of the backbone ϕ, ψ and Side-Chain χ 1 and χ 2 Dihedral Angles. Journal of Chemical Theory and Computation. 2012;8:3257–3273. doi: 10.1021/ct300400x. PubMed DOI PMC

Bolla JR, Corey RA, Sahin C, Gault J, Hummer A, Hopper JTS, Lane DP, Drew D, Allison TM, Stansfeld PJ, Robinson CV, Landreh M. A mass‐spectrometry‐based approach to distinguish annular and specific lipid binding to membrane proteins. Angewandte Chemie. 2020;132:3551–3556. doi: 10.1002/ange.201914411. PubMed DOI PMC

Bozelli JC, Aulakh SS, Epand RM. Membrane shape as determinant of protein properties. Biophysical Chemistry. 2021;273:106587. doi: 10.1016/j.bpc.2021.106587. PubMed DOI

Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. The Journal of Chemical Physics. 2007;126:014101. doi: 10.1063/1.2408420. PubMed DOI

Corey RA, Song W, Duncan AL, Ansell TB, Sansom MSP, Stansfeld PJ. Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins. Science Advances. 2021;7:eabh2217. doi: 10.1126/sciadv.abh2217. PubMed DOI PMC

Corey RA, Harrison N, Stansfeld PJ, Sansom MSP, Duncan AL. Cardiolipin, and not monolysocardiolipin, preferentially binds to the interface of complexes III and IV. Chemical Science. 2022;13:13489–13498. doi: 10.1039/d2sc04072g. PubMed DOI PMC

Corradi V, Sejdiu BI, Mesa-Galloso H, Abdizadeh H, Noskov SYu, Marrink SJ, Tieleman DP. Emerging diversity in lipid-protein interactions. Chemical Reviews. 2019;119:5775–5848. doi: 10.1021/acs.chemrev.8b00451. PubMed DOI PMC

Cournia Z, Chatzigoulas A. Allostery in membrane proteins. Current Opinion in Structural Biology. 2020;62:197–204. doi: 10.1016/j.sbi.2020.03.006. PubMed DOI

Feroz H, Kwon H, Peng J, Oh H, Ferlez B, Baker CS, Golbeck JH, Bazan GC, Zydney AL, Kumar M. Improving extraction and post-purification concentration of membrane proteins. The Analyst. 2018;143:1378–1386. doi: 10.1039/c7an01470h. PubMed DOI

Gowers R, Linke M, Barnoud J, Reddy T, Melo M, Seyler S, Domański J, Dotson D, Buchoux S, Kenney I, Beckstein O. MDAnalysis: A Python Package for the Rapid Analysis of Molecular Dynamics Simulations. SciPy Proceedings; 2016. DOI

Gupta K, Donlan JAC, Hopper JTS, Uzdavinys P, Landreh M, Struwe WB, Drew D, Baldwin AJ, Stansfeld PJ, Robinson CV. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature. 2017;541:421–424. doi: 10.1038/nature20820. PubMed DOI PMC

Hernández H, Robinson CV. Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nature Protocols. 2007;2:715–726. doi: 10.1038/nprot.2007.73. PubMed DOI

Hess B, Bekker H, Berendsen HJC, Fraaije J. LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry. 1997;18:1463–1472. doi: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H. DOI

Huang J, MacKerell AD. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. Journal of Computational Chemistry. 2013;34:2135–2145. doi: 10.1002/jcc.23354. PubMed DOI PMC

Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. Journal of Molecular Graphics. 1996;14:33–38. doi: 10.1016/0263-7855(96)00018-5. PubMed DOI

Hunte C. Specific protein-lipid interactions in membrane proteins. Biochemical Society Transactions. 2005;33:938–942. doi: 10.1042/BST20050938. PubMed DOI

Hyung SJ, Robinson CV, Ruotolo BT. Gas-phase unfolding and disassembly reveals stability differences in ligand-bound multiprotein complexes. Chemistry & Biology. 2009;16:382–390. doi: 10.1016/j.chembiol.2009.02.008. PubMed DOI

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22:2577–2637. doi: 10.1002/bip.360221211. PubMed DOI

Kimanius D, Dong L, Sharov G, Nakane T, Scheres SHW. New tools for automated cryo-EM single-particle analysis in RELION-4.0. The Biochemical Journal. 2021;478:4169–4185. doi: 10.1042/BCJ20210708. PubMed DOI PMC

Konermann L. Molecular dynamics simulations on gas-phase proteins with mobile protons: inclusion of all-atom charge solvation. The Journal of Physical Chemistry. B. 2017;121:8102–8112. doi: 10.1021/acs.jpcb.7b05703. PubMed DOI

Kroon PC, Grunewald F, Barnoud J, van Tilburg M, Souza PCT, Wassenaar TA, Marrink SJ. Martinize2 and vermouth: unified framework for topology generation. eLife. 2023;12:90627. doi: 10.7554/eLife.90627.1. DOI

Laganowsky A, Reading E, Allison TM, Ulmschneider MB, Degiacomi MT, Baldwin AJ, Robinson CV. Membrane proteins bind lipids selectively to modulate their structure and function. Nature. 2014;510:172–175. doi: 10.1038/nature13419. PubMed DOI PMC

Landreh M, Marty MT, Gault J, Robinson CV. A sliding selectivity scale for lipid binding to membrane proteins. Current Opinion in Structural Biology. 2016;39:54–60. doi: 10.1016/j.sbi.2016.04.005. PubMed DOI PMC

Landreh M, Marklund EG, Uzdavinys P, Degiacomi MT, Coincon M, Gault J, Gupta K, Liko I, Benesch JLP, Drew D, Robinson CV. Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters. Nature Communications. 2017;8:13993. doi: 10.1038/ncomms13993. PubMed DOI PMC

Larsson P, Kneiszl RC, Marklund EG. MkVsites: A tool for creating GROMACS virtual sites parameters to increase performance in all-atom molecular dynamics simulations. Journal of Computational Chemistry. 2020;41:1564–1569. doi: 10.1002/jcc.26198. PubMed DOI PMC

Levental I, Lyman E. Regulation of membrane protein structure and function by their lipid nano-environment. Nature Reviews. Molecular Cell Biology. 2023;24:107–122. doi: 10.1038/s41580-022-00524-4. PubMed DOI PMC

Lu P, Min D, DiMaio F, Wei KY, Vahey MD, Boyken SE, Chen Z, Fallas JA, Ueda G, Sheffler W, Mulligan VK, Xu W, Bowie JU, Baker D. Accurate computational design of multipass transmembrane proteins. Science. 2018;359:1042–1046. doi: 10.1126/science.aaq1739. PubMed DOI PMC

McBride JM, Polev K, Abdirasulov A, Reinharz V, Grzybowski BA, Tlusty T. AlphaFold2 can predict single-mutation effects. Physical Review Letters. 2023;131:218401. doi: 10.1103/PhysRevLett.131.218401. PubMed DOI

Michaud-Agrawal N, Denning EJ, Woolf TB, Beckstein O. MDAnalysis: A toolkit for the analysis of molecular dynamics simulations. Journal of Computational Chemistry. 2011;32:2319–2327. doi: 10.1002/jcc.21787. PubMed DOI PMC

Musatov A, Sedlák E. Role of cardiolipin in stability of integral membrane proteins. Biochimie. 2017;142:102–111. doi: 10.1016/j.biochi.2017.08.013. PubMed DOI

Nji E, Chatzikyriakidou Y, Landreh M, Drew D. An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins. Nature Communications. 2018;9:4253. doi: 10.1038/s41467-018-06702-3. PubMed DOI PMC

Páll S, Hess B. A flexible algorithm for calculating pair interactions on SIMD architectures. Computer Physics Communications. 2013;184:2641–2650. doi: 10.1016/j.cpc.2013.06.003. DOI

Palsdottir H, Hunte C. Lipids in membrane protein structures. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2004;1666:2–18. doi: 10.1016/j.bbamem.2004.06.012. PubMed DOI

Pfeiffer K, Gohil V, Stuart RA, Hunte C, Brandt U, Greenberg ML, Schägger H. Cardiolipin stabilizes respiratory chain supercomplexes. The Journal of Biological Chemistry. 2003;278:52873–52880. doi: 10.1074/jbc.M308366200. PubMed DOI

Poláchová E, Bach K, Heuten E, Stanchev S, Tichá A, Lampe P, Majer P, Langer T, Lemberg MK, Stříšovský K. Chemical blockage of the mitochondrial rhomboid protease parl by novel ketoamide inhibitors reveals its role in PINK1/Parkin-dependent mitophagy. Journal of Medicinal Chemistry. 2023;66:251–265. doi: 10.1021/acs.jmedchem.2c01092. PubMed DOI PMC

Pyle E, Kalli AC, Amillis S, Hall Z, Lau AM, Hanyaloglu AC, Diallinas G, Byrne B, Politis A. Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA. Cell Chemical Biology. 2018;25:840–848. doi: 10.1016/j.chembiol.2018.03.011. PubMed DOI PMC

Renard K, Byrne B. Insights into the role of membrane lipids in the structure, function and regulation of integral membrane proteins. International Journal of Molecular Sciences. 2021;22:9026. doi: 10.3390/ijms22169026. PubMed DOI PMC

Rimon A, Mondal R, Friedler A, Padan E. Cardiolipin is an optimal phospholipid for the assembly, stability, and proper functionality of the dimeric form of NhaA Na+/H+ Antiporter. Scientific Reports. 2019;9:17662. doi: 10.1038/s41598-019-54198-8. PubMed DOI PMC

Rohou A, Grigorieff N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. Journal of Structural Biology. 2015;192:216–221. doi: 10.1016/j.jsb.2015.08.008. PubMed DOI PMC

Schrecke S, Zhu Y, McCabe JW, Bartz M, Packianathan C, Zhao M, Zhou M, Russell D, Laganowsky A. Selective regulation of human TRAAK channels by biologically active phospholipids. Nature Chemical Biology. 2021;17:89–95. doi: 10.1038/s41589-020-00659-5. PubMed DOI PMC

Schrödinger LLC. PYMOL; 2015. https://pymol.org/

Senoo N, Chinthapalli DK, Baile MG, Golla VK, Saha B, Oluwole AO, Ogunbona OB, Saba JA, Munteanu T, Valdez Y, Whited K, Sheridan MS, Chorev D, Alder NN, May ER, Robinson CV, Claypool SM. Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier. The EMBO Journal. 2024;43:2979–3008. doi: 10.1038/s44318-024-00132-2. PubMed DOI PMC

Song W, Corey RA, Ansell TB, Cassidy CK, Horrell MR, Duncan AL, Stansfeld PJ, Sansom MSP. PyLipID: a python package for analysis of protein-lipid interactions from molecular dynamics simulations. Journal of Chemical Theory and Computation. 2022;18:1188–1201. doi: 10.1021/acs.jctc.1c00708. PubMed DOI PMC

Souza PCT, Alessandri R, Barnoud J, Thallmair S, Faustino I, Grünewald F, Patmanidis I, Abdizadeh H, Bruininks BMH, Wassenaar TA, Kroon PC, Melcr J, Nieto V, Corradi V, Khan HM, Domański J, Javanainen M, Martinez-Seara H, Reuter N, Best RB, Vattulainen I, Monticelli L, Periole X, Tieleman DP, de Vries AH, Marrink SJ. Martini 3: a general purpose force field for coarse-grained molecular dynamics. Nature Methods. 2021;18:382–388. doi: 10.1038/s41592-021-01098-3. PubMed DOI

Tichá A, Stanchev S, Škerle J, Began J, Ingr M, Švehlová K, Polovinkin L, Růžička M, Bednárová L, Hadravová R, Poláchová E, Rampírová P, Březinová J, Kašička V, Majer P, Strisovsky K. Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases. The Journal of Biological Chemistry. 2017;292:2703–2713. doi: 10.1074/jbc.M116.762849. PubMed DOI PMC

Urban S, Wolfe MS. Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity. PNAS. 2005;102:1883–1888. doi: 10.1073/pnas.0408306102. PubMed DOI PMC

Vickery ON, Stansfeld PJ. CG2AT2: an enhanced fragment-based approach for serial multi-scale molecular dynamics simulations. Journal of Chemical Theory and Computation. 2021;17:6472–6482. doi: 10.1021/acs.jctc.1c00295. PubMed DOI PMC

Vinothkumar KR. Structure of rhomboid protease in a lipid environment. Journal of Molecular Biology. 2011;407:232–247. doi: 10.1016/j.jmb.2011.01.029. PubMed DOI PMC

Wang Y, Ha Y. Open-cap conformation of intramembrane protease GlpG. PNAS. 2007;104:2098–2102. doi: 10.1073/pnas.0611080104. PubMed DOI PMC

Wassenaar TA, Ingólfsson HI, Böckmann RA, Tieleman DP, Marrink SJ. Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations. Journal of Chemical Theory and Computation. 2015;11:2144–2155. doi: 10.1021/acs.jctc.5b00209. PubMed DOI

Weikum J, van Dyck JF, Subramani S, Klebl DP, Storflor M, Muench SP, Abel S, Sobott F, Morth JP. The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2024;1871:119614. doi: 10.1016/j.bbamcr.2023.119614. PubMed DOI

Yen HY, Abramsson ML, Agasid MT, Lama D, Gault J, Liko I, Kaldmäe M, Saluri M, Qureshi AA, Suades A, Drew D, Degiacomi MT, Marklund EG, Allison TM, Robinson CV, Landreh M. Electrospray ionization of native membrane proteins proceeds via a charge equilibration step. RSC Advances. 2022;12:9671–9680. doi: 10.1039/d2ra01282k. PubMed DOI PMC

Zivanov J, Nakane T, Scheres SHW. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ. 2019;6:5–17. doi: 10.1107/S205225251801463X. PubMed DOI PMC