Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts, together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and molecular dynamics simulations prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role-it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

Department of Chemistry Faculty of Science University of South Bohemia in České Budějovice 370 05 České Budějovice Czech Republic

Institute of Biotechnology University of Helsinki 00790 Helsinki Finland

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nam 2 166 10 Prague 6 Czech Republic

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 2155 3 182 23 Prague 8 Czech Republic

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Halling DB, Liebeskind BJ, Hall AW, Aldrich RW. 2016. Conserved properties of individual Ca2+-binding sites in calmodulin. Proc. Natl Acad. Sci. USA 113 , E1216–E1225. (10.1073/pnas.1600385113) PubMed DOI PMC

Jurado LA, Chockalingam PS, Jarrett HS. 1999. Apocalmodulin. Physiol. Rev. 79 , 661–682. (10.1152/physrev.1999.79.3.661) PubMed DOI

Tidow H, Nissen P. 2013. Structural diversity of calmodulin binding to its target sites. FEBS J. 280 , 5551–5565. (10.1111/febs.12296) PubMed DOI

Kopeikina-Tsiboukidou L, Deliconstantinos G. 1989. Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes. Neurochem. Res. 14 , 119–127. (10.1007/BF00969626) PubMed DOI

Yoshida M, Tanaka J, Tamura J, Fujita K, Kasamatsu T, Kohmoto M, Tobe T. 1993. Altered fluidity of liver plasma membranes following partial hepatectomy in rats. J. Surg. Res. 55 , 390–396. (10.1006/jsre.1993.1158) PubMed DOI

Kopeikina L, Degiannis E, Villiotou V, Stavridis I. 1997. Calmodulin-related changes in microsomal membrane fluidity during liver regeneration. J. Surg. Res. 67 , 155–162. (10.1006/jsre.1996.4975) PubMed DOI

Chamberlain SG, Gohlke A, Shafiq A, Squires IJ, Owen D, Mott HR. 2021. Calmodulin extracts the Ras family protein RalA from lipid bilayers by engagement with two membrane-targeting motifs. Proc. Natl Acad. Sci. USA 118 , e2104219118. (10.1073/pnas.2104219118) PubMed DOI PMC

Kovacs E, Harmat V, Tóth J, Vértessy BG, Módos K, Kardos J, Liliom K. 2010. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. FASEB J. 24 , 3829–3839. (10.1096/fj.10-155614) PubMed DOI PMC

Kovacs E, Tóth J, Vértessy BG, Liliom K. 2010. Dissociation of calmodulin-target peptide complexes by the lipid mediator sphingosylphosphorylcholine: implications in calcium signaling. J. Biol. Chem. 285 , 1799–1808. (10.1074/jbc.M109.053116) PubMed DOI PMC

Grant BMM, Enomoto M, Back SI, Lee KY, Gebregiworgis T, Ishiyama N, Ikura M, Marshall CB. 2020. Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety. Sci. Signal. 13 , 625. (10.1126/scisignal.aaz0344) PubMed DOI

Maruyama Y, Ueno S, Morita M, Hayashi F, Maekawa S. 2018. Inhibitory effect of several sphingolipid metabolites on calcineurin. Neurosci. Lett. 673 , 132–135. (10.1016/j.neulet.2018.03.010) PubMed DOI

Homola J. 2008. Surface plasmon resonance sensors for detection of chemical and biological species. Chem. Rev. 108 , 462–493. (10.1021/cr068107d) PubMed DOI

Rich RL, Myszka DG. 2000. Advances in surface plasmon resonance biosensor analysis. Curr. Opin. Biotechnol. 11 , 54–61. (10.1016/s0958-1669(99)00054-3) PubMed DOI

Jung LS, Campbell CT, Chinowsky TM, Mar MN, Yee SS. 1998. Quantitative interpretation of the response of surface plasmon resonance sensors to adsorbed films. Langmuir 14 , 5636–5648. (10.1021/la971228b) DOI

Viitala T, Liang HM, Gupta M, Zwinger T, Yliperttula M, Bunker A. 2012. Fluid dynamics modeling for synchronizing surface plasmon resonance and quartz crystal microbalance as tools for biomolecular and targeted drug delivery studies. J. Colloid Interface Sci. 378 , 251–259. (10.1016/j.jcis.2012.04.012) PubMed DOI

Besenicar M, Macek P, Lakey JH, Anderluh G. 2006. Surface plasmon resonance in protein-membrane interactions. Chem. Phys. Lipids 141 , 169–178. (10.1016/j.chemphyslip.2006.02.010) PubMed DOI

Zheng WF, Wang LJ, Hong YK, Sha YL. 2009. PrP106–126 peptide disrupts lipid membranes: influence of C-terminal amidation. Biochem. Biophys. Res. Commun. 379 , 298–303. (10.1016/j.bbrc.2008.12.049) PubMed DOI

Almeida I, Marquês JT, Liu W, Niu Y, de Almeida RFM, Jin G, Viana AS. 2015. Phospholipid/cholesterol/decanethiol mixtures for direct assembly of immunosensing interfaces. Colloids Surf. B 136 , 997–1003. (10.1016/j.colsurfb.2015.10.048) PubMed DOI

Stace CL, Ktistakis NT. 2006. Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1761 , 913–926. (10.1016/j.bbalip.2006.03.006) PubMed DOI

Kerr D, Tietjen GT, Gong ZL, Tajkhorshid E, Adams EJ, Lee KYC. 2018. Sensitivity of peripheral membrane proteins to the membrane context: a case study of phosphatidylserine and the TIM proteins. Biochim. Biophys. Acta Biomembr. 1860 , 2126–2133. (10.1016/j.bbamem.2018.06.010) PubMed DOI PMC

Dymond MK. 2021. Lipid monolayer spontaneous curvatures: a collection of published values. Chem. Phys. Lipids 239 , 105117. (10.1016/j.chemphyslip.2021.105117) PubMed DOI

Parasassi T, De Stasio G, d’Ubaldo A, Gratton E. 1990. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys. J. 57 , 1179–1186. (10.1016/S0006-3495(90)82637-0) PubMed DOI PMC

Amaro M, Šachl R, Jurkiewicz P, Coutinho A, Prieto M, Hof M. 2014. Time-resolved fluorescence in lipid bilayers: selected applications and advantages over steady state. Biophys. J. 107 , 2751–2760. (10.1016/j.bpj.2014.10.058) PubMed DOI PMC

Bagatolli LA. 2012. Laurdan fluorescence properties in membranes: a journey from the fluorometer to the microscope. In Fluorescent methods to study biological membranes (eds Mély Y, Duportail G), pp. 3–35. Berlin, Germany: Springer. (10.1007/4243_2012_42) DOI

Hutterer R, Schneider FW, Hermens WT, Wagenvoord R, Hof M. 1998. Binding of prothrombin and its fragment 1 to phospholipid membranes studied by the solvent relaxation technique. Biochim. Biophys. Acta Biomemb. 1414 , 155–164. (10.1016/S0005-2736(98)00163-1) PubMed DOI

Su Q, Vogt S, Nöll G. 2018. Langmuir analysis of the binding affinity and kinetics for surface tethered duplex DNA and a ligand-apoprotein complex. Langmuir 34 , 14738–14748. (10.1021/acs.langmuir.7b04347) PubMed DOI

Olšinová M, Jurkiewicz P, Kishko I, Sýkora J, Sabó J, Hof M, Cwiklik L, Cebecauer M. 2018. Roughness of transmembrane helices reduces lipid membrane dynamics. iScience 10 , 87–97. (10.1016/j.isci.2018.11.026) PubMed DOI PMC

Sheynis T, Sykora J, Benda A, Kolusheva S, Hof M, Jelinek R. 2003. Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies. Eur. J. Biochem. 270 , 4478–4487. (10.1046/j.1432-1033.2003.03840.x) PubMed DOI

Rieber K, Sýkora J, Olzyńska A, Jelinek R, Cevc G, Hof M. 2007. The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes. Biochim. Biophys. Acta 1768 , 1050–1058. (10.1016/j.bbamem.2006.12.018) PubMed DOI

Macháň R, Jurkiewicz P, Olżyńska A, Olšinová M, Cebecauer M, Marquette A, Bechinger B, Hof M. 2014. Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH(4). Langmuir 30 , 6171–6179. (10.1021/la5006314) PubMed DOI

Duboué-Dijon E, Javanainen M, Delcroix P, Jungwirth P, Martinez-Seara H. 2020. A practical guide to biologically relevant molecular simulations with charge scaling for electronic polarization. J. Chem. Phys. 153 , 050901. (10.1063/5.0017775) PubMed DOI

Melcrová A, Pokorna S, Pullanchery S, Kohagen M, Jurkiewicz P, Hof M, Jungwirth P, Cremer PS, Cwiklik L. 2016. The complex nature of calcium cation interactions with phospholipid bilayers. Sci. Rep. 6 , 38035. (10.1038/srep38035) PubMed DOI PMC

Davletov B, Perisic O, Williams RL. 1998. Calcium-dependent membrane penetration is a hallmark of the C2 domain of cytosolic phospholipase A2 whereas the C2A domain of synaptotagmin binds membranes electrostatically. J. Biol. Chem. 273 , 19093–19096. (10.1074/jbc.273.30.19093) PubMed DOI

Huang BX, Akbar M, Kevala K, Kim HY. 2011. Phosphatidylserine is a critical modulator for Akt activation. J. Cell Biol. 192 , 979–992. (10.1083/jcb.201005100) PubMed DOI PMC

Wolny M, et al. . 2011. Key amino acid residues of ankyrin-sensitive phosphatidylethanolamine/phosphatidylcholine-lipid binding site of βI-spectrin. PLoS ONE 6 , e21538. (10.1371/journal.pone.0021538) PubMed DOI PMC

Bazzi MD, Youakim A, Nelsestuen GL. 1992. Importance of phosphatidylethanolamine for association of protein-kinase-C and other cytoplasmic proteins with membranes. Biochemistry 31 , 1125–1134. (10.1021/bi00119a022) PubMed DOI

Medfisch SM, Muehl EM, Morrissey JH, Bailey RC. 2020. Phosphatidylethanolamine-phosphatidylserine binding synergy of seven coagulation factors revealed using nanodisc arrays on silicon photonic sensors. Sci. Rep. 10 , 17407. (10.1038/s41598-020-73647-3) PubMed DOI PMC

Vance JE, Tasseva G. 2013. Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1831 , 543–554. (10.1016/j.bbalip.2012.08.016) PubMed DOI

Iwamoto K, Hayakawa T, Murate M, Makino A, Ito K, Fujisawa T, Kobayashi T. 2007. Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin. Biophys. J. 93 , 1608–1619. (10.1529/biophysj.106.101584) PubMed DOI PMC

Makino A, et al. . 2003. Cinnamycin (Ro 09-0198) promotes cell binding and toxicity by inducing transbilayer lipid movement. J. Biol. Chem. 278 , 3204–3209. (10.1074/jbc.M210347200) PubMed DOI

Sperlich B, Kapoor S, Waldmann H, Winter R, Weise K. 2016. Regulation of K-Ras4B membrane binding by calmodulin. Biophys. J. 111 , 113–122. (10.1016/j.bpj.2016.05.042) PubMed DOI PMC

Matsubara M, Nakatsu T, Kato H, Taniguchi H. 2004. Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca2+/calmodulin. EMBO J. 23 , 712–718. (10.1038/sj.emboj.7600093) PubMed DOI PMC

Scollo F, Evci H, Jurkiewicz P. 2024. Data for: Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes? Zenodo (10.5281/zenodo.10843995) PubMed DOI PMC

Scollo F, Evci H, Jurkiewicz P. 2024. Additional data for: can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes? zenodo (10.5281/zenodo.12795857) PubMed DOI PMC

Tempra C, Martinez-Seara H. 2024. Data for: Apo-Calmodulin interacting with membranes. Zenodo (10.5281/zenodo.10727235) DOI

Tempra C, Martinez-Seara H. 2024. Data for: Holo-Calmodulin interacting with membranes. Zenodo (10.5281/zenodo.10727430) DOI

Tempra C, Martinez-Seara H. 2024. Data for: Holo-Calmodulin interacting with membranes with extra CaCl2 in the environment. Zenodo (10.5281/zenodo.10727572) DOI

Scollo F, et al. . 2024. Data from: Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes? Figshare (10.6084/m9.figshare.c.7430650) PubMed DOI PMC

Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?