{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:58:32Z","timestamp":1792807112121,"version":"4.5.0"},"reference-count":47,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2020,6,27]],"date-time":"2020-06-27T00:00:00Z","timestamp":1593216000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P20 GM103554"],"award-info":[{"award-number":["P20 GM103554"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P41GM103712"],"award-info":[{"award-number":["P41GM103712"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30DA035778"],"award-info":[{"award-number":["P30DA035778"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"University of Nevada at Reno","award":["Nevada INBRE P20 GM103440"],"award-info":[{"award-number":["Nevada INBRE P20 GM103440"]}],"id":[{"id":"https:\/\/ror.org\/01keh0577","id-type":"ROR","asserted-by":"crossref"}]},{"name":"University of Nevada at Reno","award":["1200-119-1440"],"award-info":[{"award-number":["1200-119-1440"]}],"id":[{"id":"https:\/\/ror.org\/01keh0577","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Cardiotoxin CTII from Naja oxiana cobra venom translocates to the intermembrane space (IMS) of mitochondria to disrupt the structure and function of the inner mitochondrial membrane. At low concentrations, CTII facilitates ATP-synthase activity, presumably via the formation of non-bilayer, immobilized phospholipids that are critical in modulating ATP-synthase activity. In this study, we investigated the effects of another cardiotoxin CTI from Naja oxiana cobra venom on the structure of mitochondrial membranes and on mitochondrial-derived ATP synthesis. By employing robust biophysical methods including 31P-NMR and 1H-NMR spectroscopy, we analyzed the effects of CTI and CTII on phospholipid packing and dynamics in model phosphatidylcholine (PC) membranes enriched with 2.5 and 5.0 mol% of cardiolipin (CL), a phospholipid composition that mimics that in the outer mitochondrial membrane (OMM). These experiments revealed that CTII converted a higher percentage of bilayer phospholipids to a non-bilayer and immobilized state and both cardiotoxins utilized CL and PC molecules to form non-bilayer structures. Furthermore, in order to gain further understanding on how cardiotoxins bind to mitochondrial membranes, we employed molecular dynamics (MD) and molecular docking simulations to investigate the molecular mechanisms by which CTII and CTI interactively bind with an in silico phospholipid membrane that models the composition similar to the OMM. In brief, MD studies suggest that CTII utilized the N-terminal region to embed the phospholipid bilayer more avidly in a horizontal orientation with respect to the lipid bilayer and thereby penetrate at a faster rate compared with CTI. Molecular dynamics along with the Autodock studies identified critical amino acid residues on the molecular surfaces of CTII and CTI that facilitated the long-range and short-range interactions of cardiotoxins with CL and PC. Based on our compiled data and our published findings, we provide a conceptual model that explains a molecular mechanism by which snake venom cardiotoxins, including CTI and CTII, interact with mitochondrial membranes to alter the mitochondrial membrane structure to either upregulate ATP-synthase activity or disrupt mitochondrial function.<\/jats:p>","DOI":"10.3390\/toxins12070425","type":"journal-article","created":{"date-parts":[[2020,6,30]],"date-time":"2020-06-30T14:29:39Z","timestamp":1593527379000},"page":"425","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Molecular Mechanism by Which Cobra Venom Cardiotoxins Interact with the Outer Mitochondrial Membrane"],"prefix":"10.3390","volume":"12","author":[{"given":"Feng","family":"Li","sequence":"first","affiliation":[{"name":"STEM Program, Science Department, Chaoyang KaiWen Academy, Yard 46, 3rd Baoquan Street, Chaoyang District, Beijing 100018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Indira H.","family":"Shrivastava","sequence":"additional","affiliation":[{"name":"Department of Computational and System Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA"},{"name":"Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15260, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Hanlon","sequence":"additional","affiliation":[{"name":"STEM Program, Science Department, Chaoyang KaiWen Academy, Yard 46, 3rd Baoquan Street, Chaoyang District, Beijing 100018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruben K.","family":"Dagda","sequence":"additional","affiliation":[{"name":"Reno School of Medicine, Department of Pharmacology, University of Nevada, Reno, NV 89557, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5097-3973","authenticated-orcid":false,"given":"Edward S.","family":"Gasanoff","sequence":"additional","affiliation":[{"name":"STEM Program, Science Department, Chaoyang KaiWen Academy, Yard 46, 3rd Baoquan Street, Chaoyang District, Beijing 100018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Gasanov, S.E., Shrivastava, I.H., Israilov, F.S., Kim, A.A., Rylova, K.A., Zhang, B., and Dagda, R.K. (2015). Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0129248"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1016\/j.plipres.2013.07.002","article-title":"Lipids of mitochondria","volume":"52","author":"Horvath","year":"2013","journal-title":"Prog. Lipid Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"586","DOI":"10.1016\/j.bbamem.2017.11.014","article-title":"Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity","volume":"1860","author":"Gasanov","year":"2018","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1134\/S0006350916040084","article-title":"The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes","volume":"61","author":"Gasanov","year":"2016","journal-title":"Biophysics"},{"key":"ref_5","first-page":"705","article-title":"Possible role of non-bilayer structures in regulating the activity of ATP synthase in mitochondria","volume":"61","author":"Gasanov","year":"2016","journal-title":"Biofizika"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Zhang, B., Li, F., Chen, Z., Srivastava, I.H., Gasanoff, E.S., and Dagda, R.K. (2019). Naja mosambica mossambica cobra cardiotoxin targets mitochondria to disrupt mitochondrial membrane structure and function. Toxins, 11.","DOI":"10.3390\/toxins11030152"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1016\/0005-2736(80)90466-6","article-title":"Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A 31P NMR study","volume":"600","author":"Cullis","year":"1980","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_8","unstructured":"De Kruijff, B., Cullis, P., Verkleij, A., Hope, M., Van Echteld, C., Taraschi, T., Van Hoogevest, P., Killian, J., Rietveld, A., and Van der Steen, A. (1985). Modulation of lipid polymorphism by lipid-protein interactions. Prog. Protein Lipid Interact., 89\u2013142."},{"key":"ref_9","first-page":"459","article-title":"Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes","volume":"8","author":"Aripov","year":"1989","journal-title":"Gen. Physiol. Biophys."},{"key":"ref_10","first-page":"1045","article-title":"Formation of nonbilayer structures in phospholipid membranes induced by cationic polypeptides","volume":"7","author":"Gasanov","year":"1990","journal-title":"Biol. Membr."},{"key":"ref_11","first-page":"42","article-title":"The fusogenic properties of the cytotoxins of cobra venom in a model membrane system","volume":"2","author":"Gasanov","year":"1990","journal-title":"Nauchnye Dokl. Vysshei Shkoly Biol. Nauki"},{"key":"ref_12","first-page":"958","article-title":"Intermembrane exchange of lipids induced by cobra venom cytotoxins","volume":"35","author":"Gasanov","year":"1990","journal-title":"Biofizika"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1006\/abbi.1993.1157","article-title":"Modification of phospholipid membrane structure by the plant toxic peptide Pyrularia thionin","volume":"301","author":"Gasanov","year":"1993","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1007\/s002329900165","article-title":"Cobra venom cytotoxin free of phospholipase A2 and its effect on model membranes and T leukemia cells","volume":"155","author":"Gasanov","year":"1997","journal-title":"J. Membr. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1007\/s10858-013-9750-x","article-title":"An overview of tools for the validation of protein NMR structures","volume":"58","author":"Vuister","year":"2014","journal-title":"J. Biomol. NMR"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0304-4157(85)90002-4","article-title":"Lipids of mitochondria","volume":"822","author":"Daum","year":"1985","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.compbiolchem.2008.07.018","article-title":"Steered molecular dynamics simulations of cobra cytotoxin interaction with zwitterionic lipid bilayer: No penetration of loop tips into membranes","volume":"33","author":"Levtsova","year":"2009","journal-title":"Comput. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Konshina, A.G., Boldyrev, I.A., Utkin, Y.N., Omel\u2019kov, A.V., and Efremov, R.G. (2011). Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0019064"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"88","DOI":"10.32607\/20758251-2010-2-2-88-95","article-title":"Anionic lipids: Determinants of binding cytotoxins from snake venom on the surface of cell membranes","volume":"2","author":"Konshina","year":"2010","journal-title":"Acta Nat."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/0014-5793(74)80462-X","article-title":"The isolation and sequence determination of a cytotoxin from the venom of the Middle-Asian cobra Naja naja oxiana","volume":"48","author":"Grishin","year":"1974","journal-title":"FEBS Lett."},{"key":"ref_21","first-page":"310","article-title":"Isolation and characteristics of a \u201cdirect\u201d haemolytic factor of Central Asian cobra venom","volume":"39","author":"Yukelson","year":"1974","journal-title":"Biokhimiya"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1042\/BJ20041892","article-title":"Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage","volume":"390","author":"Feofanov","year":"2005","journal-title":"Biochem. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1142\/S0192415X13500456","article-title":"The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release","volume":"41","author":"Wu","year":"2013","journal-title":"Am. J. Chin. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1016\/j.toxicon.2007.11.024","article-title":"Taiwan cobra cardiotoxins induce apoptotic death of human neuroblastoma SK-N-SH cells mediated by reactive oxygen species generation and mitochondrial depolarization","volume":"51","author":"Chen","year":"2008","journal-title":"Toxicon"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1000181","DOI":"10.4172\/2161-0495.1000181","article-title":"Snake venom cytotoxins, phospholipase A2s, and Zn2+-dependent metalloproteinases: Mechanisms of action and pharmacological relevance","volume":"4","author":"Gasanov","year":"2014","journal-title":"J. Clin. Toxicol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11402","DOI":"10.1021\/jp801825k","article-title":"Pore structure, thinning effect, and lateral diffusive dynamics of oriented lipid membranes interacting with antimicrobial peptide protegrin-1: 31P and 2H solid-state NMR study","volume":"112","author":"Wi","year":"2008","journal-title":"J. Phys. Chem. B"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3169","DOI":"10.1016\/j.febslet.2005.05.006","article-title":"Amphiphilic beta-sheet cobra cardiotoxin targets mitochondria and disrupts its network","volume":"579","author":"Wang","year":"2005","journal-title":"FEBS Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/j.toxicon.2008.06.013","article-title":"Involvement of mitochondrial alteration and reactive oxygen species generation in Taiwan cobra cardiotoxin-induced apoptotic death of human neuroblastoma SK-N-SH cells","volume":"52","author":"Chen","year":"2008","journal-title":"Toxicon"},{"key":"ref_29","first-page":"728","article-title":"Interaction of cobra venom cytotoxin with oriented phospholipid multi-bilayers","volume":"288","author":"Aripov","year":"1986","journal-title":"Dokl. Akad. Nauk SSSR"},{"key":"ref_30","first-page":"275","article-title":"Modulation of phospholipase A2 activity by membrane-active peptides on liposomes of different phospholipid composition","volume":"13","author":"Gasanov","year":"1994","journal-title":"Gen. Physiol. Biophys."},{"key":"ref_31","first-page":"107","article-title":"Phospholipase A2 and cobra venom cytotoxin Vc5 interactions and membrane structure","volume":"14","author":"Gasanov","year":"1995","journal-title":"Gen. Physiol. Biophys."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/BF00700663","article-title":"An asymmetric enlargement of the monolayer surfaces mechanism of membrane fusion","volume":"19","author":"Gasanov","year":"1994","journal-title":"J. Biol. Phys."},{"key":"ref_33","unstructured":"Gasanov, S.E., Aripov, T.F., and Gasanov, E.E. (1988). Study on Structure of Phospholipid Membranes Modified by Membrane-Active Polypeptides, Nuclear Physics Institute UzSSR Academy of Sciences."},{"key":"ref_34","first-page":"1812","article-title":"Mitochondrial proteolipids","volume":"58","author":"Segal","year":"1993","journal-title":"Biokhimiia"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2036","DOI":"10.1016\/S0021-9258(19)41680-3","article-title":"The relationship between the bovine heart mitochondrial adenosine triphosphatase, lipophilic compounds, and oligomycin","volume":"250","author":"Cunningham","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Rogers, G.W., Brand, M.D., Petrosyan, S., Ashok, D., Elorza, A.A., Ferrick, D.A., and Murphy, A.N. (2011). High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0021746"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"R1259","DOI":"10.1152\/ajpregu.00264.2003","article-title":"Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction","volume":"285","author":"Drew","year":"2003","journal-title":"Am. J. Phys. Regul. Integr. Comp. Physiol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Smith, I.C., and Ekiel, I.H. (1984). Phosphorus-31 NMR of Phospholipids in Membranes. Phosphorous-31 NMR: Principles and Applications, Academic Press.","DOI":"10.1016\/B978-0-08-091841-9.50020-5"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1042\/bst0240274","article-title":"ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling","volume":"24","author":"Peitsch","year":"1996","journal-title":"Biochem. Soc. Trans."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1093\/bioinformatics\/btt055","article-title":"GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit","volume":"29","author":"Pronk","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1007\/s00249-004-0448-6","article-title":"Validation of the 53A6 GROMOS force field","volume":"34","author":"Oostenbrink","year":"2005","journal-title":"Eur. Biophys. J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H","article-title":"LINCS: A linear constraint solver for molecular simulations","volume":"18","author":"Hess","year":"1997","journal-title":"J. Comput. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle Mesh Ewald\u2014An N.Log(N) Method for Ewald Sums in Large Systems","volume":"98","author":"Darden","year":"1993","journal-title":"J. Chem. Phys."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1007\/BF00355047","article-title":"PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules","volume":"10","author":"Bywater","year":"1996","journal-title":"J. Comput. Aided Mol. Des."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"9220","DOI":"10.1021\/jacs.9b13450","article-title":"Charge transfer and chemo-mechanical coupling in respiratory complex I","volume":"142","author":"Gupta","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1186\/1758-2946-4-17","article-title":"Avogadro: An advanced semantic chemical editor, visualization, and analysis platform","volume":"4","author":"Hanwell","year":"2012","journal-title":"J. Cheminform."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/12\/7\/425\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:43:45Z","timestamp":1760175825000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/12\/7\/425"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,27]]},"references-count":47,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2020,7]]}},"alternative-id":["toxins12070425"],"URL":"https:\/\/doi.org\/10.3390\/toxins12070425","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,27]]}}}