{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD065966/J1000.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD065966/J1000_mzML.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD065966/J1000_ragPipe_Results.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD065966/J1000_raw.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["pnovac@biomed.cas.cz"],"submitter":["Sascha Vatic"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"disease":["Bordetella Parapertussis Whooping Cough"],"software":[""],"submitter_keywords":["Rtx toxin","Adenylate cyclase toxin","Folding","Calcium binding sites"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD065966"],"sample_protocol":["The acylation status of proteins was analyzed by liquid chromatography-mass spectrometry (LC-MS) as described earlier (Scientific Reports 11, 19814). Briefly, purified CyaA and HlyA toxins and their mutant variants in 8 M urea were normalized to the final concentration of 0,5 mg/mL, and the buffer conditions of 4 M urea, 10% acetonitrile (v/v), 50 mM N-ethylmorpholine / acetate, pH 8,30, and subsequently digested for 2 h at 30 °C with LysC (1/40 enzyme: substrate ratio; Lysyl Endopeptidase, FUJIFILM Wako, Richmond, VA, U.S.A.) and an additional 4 h at 30 °C with trypsin (1/40 enzyme: substrate ratio; Sequencing Grade Modified Trypsin, Promega, Madison, WI, U.S.A.). Digestion was quenched with trifluoroacetic acid (Merck, Rahway, NJ, U.S.A.) to reach a 0.1% final concentration.Resulting peptides were diluted 1:1 in 0.1 % FA (v/v), and 1.25 μg equivalents were loaded on the C18 trap column (20 x 0.30 mm, Luna Omega 5 μm Polar C18 100 Å, Phenomenex, Torrance, CA, U.S.A.) and subsequently separated on an analytical column (150 x 0.3mm, Luna Omega 3 μm Polar C18 100 Å, Phenomenex, Torrance, CA, U.S.A.). Chromatographic separation was carried out on Agilent 1290 binary pump and 1260 linear pump (Agilent Technologies, Santa Clara, CA, U.S.A.), using linear gradient of 5-65 % solution B (70 % acetonitrile, 25 % isopropanol, 0.16 % formic acid) over solution A (2,5 % acetonitrile, 2,5 % isopropanol, 0.20 % formic acid) at a flow rate of 10 μL/min over 34 min and linear gradient 65-95 % for an additional 1 min. LC-MS/MS data were acquired on a maXis I Q-ToF instrument (Bruker Daltonics, Billerica, MA, U.S.A.) using a data-dependent auto-MS/MS method in positive mode, selecting the 5 most abundant precursor ions with a cycle time of 3.0 s, mass range from m/z 200 to 1700, and exclusion release of 19 s."],"repository":["pride"],"modification":[""],"quantification_method":[""],"data_protocol":["All runs were converted from Bruker .d to mzML format using Proteo Wizard MSConvert (v3.0.25178-521213f (automated build)) [https://doi.org/10.1038/nbt.2377], and processed with custom mass offsets with MSFragger (v4.3), and IonQuant (v1.11.11) in FragPipe platform (v23.0) [https://doi.org/10.1016/j.mcpro.2021.100077] against a custom database containing CyaA and HlyA wild-type and mutant variants, and standard contaminant sequences (cRAP.fasta) with 1% FDR value. The FragPipe settings were as follows: K/R protease specificity, two missed cleavages, no fixed modification and stearoylation, oleoylation, linoleoylation, linolenoylation, palmitoleoylation, juniperoylation, palmitoylation, hydroxymyristoylation, myristoylation, myristoleoylation, variable modifications. Quantitation was done based on the intensity of the first isotope at the maximum of the EIC in Bruker Compass DataAnalysis software v6.1(Bruker Daltonics, Billerica, MA, U.S.A.). All files are available on PRIDE."],"omics_type":["Proteomics"],"labhead":["Petr Novak"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["The Institute of Microbiology of the Czech Academy of Sciences Head of Group Protein Structure Characterization by Advanced Mass Spectrometry"],"species":["Escherichia Coli","Bacteria","Bordetella Pertussis Nbrc 107857"],"publication":["10.1016/J.JBC.2026.113079"],"submitter_mail":["sascha.vatic@biomed.cas.cz"],"submitter_affiliation":["Biocev MBU CAS"],"submitter_country":["Czechia"],"additional_accession":[]},"is_claimable":false,"name":"Calcium-loaded acylated segment controls the membrane penetration capacity of repeats-in-toxin cytolysins","description":"Loading of calcium ions into the numerous carboxy-terminus-proximal binding sites in the Repeats in ToXin (RTX) domains drives the cooperative and vectorial folding of RTX β-roll structures involved in cell binding and membrane penetration of RTX cytolysins. Two additional binding sites for calcium ions, coordinated by the side chains of residues D880, D918, and N936, were identified in the structure of the acylated N-terminal cap of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA). Here, we show that this calcium-binding structure plays a key role in membrane insertion of the toxin. An N936L residue substitution did not impact toxin acylation or CR3 receptor binding but disrupted the calcium-dependent fold of the acylated segment and ablated the membrane penetration capacity of the toxin. Similarly, substitution of the corresponding D639 residue of Escherichia coli α-hemolysin (HlyA) abolished its cytolytic capacity. Moreover, disruption of the β-turn structures in the calcium binding sites of the acylated segment of CyaA (G934L) and HlyA (G637L) strongly impaired the cytotoxic capacities of both toxins. On the contrary, a D880L substitution yielded a toxin with an enhanced CR3-independent cell penetration and pore-forming capacity. Hydrogen/deuterium exchange probing revealed that the D880L substitution altered the fold of the acylated segment and stabilized the interaction of its two acylated β-hairpins. Hence, loading the calcium-binding sites in the acylated segment controls its structure and rules the interaction and functional cooperation of its two acylated β-hairpins that initiate penetration of the CyaA polypeptide into the cell membrane.","dates":{"publication":"2026-04-28","submission":"2025-07-09"},"accession":"PXD065966","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:5722","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:1211601","NEWT:876138","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:10029","NEWT:1000589","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:499175","NEWT:476272","NEWT:3747","NEWT:367830","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:28038","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:4081","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:2829","NEWT:216599","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:260707","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","NCBITaxon:6073","NEWT:884204","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:38783","NEWT:8727","NEWT:4006","NEWT:8726","NEWT:6426","NEWT:6669","NEWT:10090","NEWT:4120","NEWT:51515","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","NEWT:5334","NEWT:37334","NEWT:382352","NCBITaxon:10359","NEWT:242619","NEWT:632957","NEWT:373995","NEWT:544404","NEWT:9925","NEWT:8839","NEWT:4232","NEWT:2758385","NEWT:4113","NEWT:837","NEWT:11298","NEWT:171101","NEWT:714","NEWT:421932","NEWT:196627","NEWT:5691","NEWT:48479","NEWT:627025","NEWT:1097677","NEWT:61674","NEWT:1117957","NEWT:9913","NEWT:4100","NEWT:1076","NEWT:6763","NEWT:803","NEWT:29722","NEWT:380394","NEWT:1692259","NEWT:180066","NEWT:135588","NEWT:1843183","NEWT:58002","NEWT:3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