{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/Complex_II_RAB5.txt","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/checksum.txt"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/Complex_II_RAB5.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/Relative_Fractional_Uptake_plots.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/Complex_II.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061277/PLGS_SEARCH.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["rlw@mrc-lmb.cam.ac.uk"],"submitter":["Maxime Bourguet"],"technology_type":["HDMSE","Mass Spectrometry"],"software":["Not available"],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD061277"],"tissue":["Cell Culture"],"sample_protocol":["HDX-MS experiments were carried out as follows: Complex II (48 μM) alone or in the presence of 90 μM RAB5A-Q79L-C19S-C63S-GTP were pre-incubated for 30 min on ice. Complex II alone sample was then exposed for different deuteration times (3 sec on ice, 3, 30, 300, and 3000 sec at room temperaturert) to D2O only buffer (20 mM Hepes pD8, 300 mM NaCl) to yield solution that was 5 μM Complex II and D2O at 83% final concentration. Complex II +RAB5 A sample was exposed for the same deuteration times to a D2O+RAB5 A buffer (20 mM Hepes pD8, 300 mM NaCl, 90 μM RAB5A) to yield solution that was and 5 μM Complex II +90 μM RAB5A and D2O at 83% final concentration. Samples were then immediately flash-frozen in liquid nitrogen and stored at –80 °C until analysis. Prior to LC-MS analysis, samples were quickly thawed and injected on a HDX Manager coupled to an Acquity UPLC M-Class system (Waters) set at 0.1  °C. Samples were then digested online at 15  °C using a 20 mm x 2.0 mm home-made pepsin column (Pepsin from Thermo Fisher Scientific, 20343 and hardware from Upchurch Scientific, C130-B) and loaded on a UPLC pre-column (ACQUITY UPLC BEH C18 VanGuard pre-column, 2.1  mm I.D. ×5  mm, 1.7  μm particle diameter, Waters, 186003975) for 2 min at 100 μL/min. Digested peptides were then eluted over 20 min from the pre-column onto an ACQUITY UPLC BEH C18 column (1.0  mm I.D. × 100  mm, 1.7  μm particle diameter, Waters, 186002346) using a 5–43% gradient of acetonitrile (Romil, H050), 0.1% formic acid. MS data were acquired using a Synapt G2Si-HDMS (Waters) with electrospray ionizsation, using the data-independent acquisition mode (HDMSE) over an m/z range of 50–2000 and a 100  fmol/μL Glu-FibrinoPeptide solution was used for lock-mass correction and calibration. The following parameters were used during the acquisition: capillary voltage, 3 kV; sampling cone voltage, 40 V; source temperature, 90 °C; desolvation gas, 150 °C and 650 L.h−1; scan time, 0.3 s; trap collision energy ramp, 20–45 eV."],"repository":["Pride"],"modification":[""],"quantification_method":[""],"data_protocol":["Peptide identification was performed using ProteinLynx Global Server 3.0.1 (PLGS, Waters) with a home-made protein sequence library containing VPS34, VPS15, UVRAG, BECLIN1, RAB5A-Q79L-C19S-C63S-GTP, and pepsin sequences, with peptide and fragment tolerances set automatically by PLGS. Peptides were filtered using HDExaminer 3.4.0 (Trajan Scientific and Medical) with a minimum fragment per amino acid of 0.2, a minimum intensity of 1000, a length between 5 and 25 residues, a minimum PLGS score of 6.62, and an MH+ tolerance of 5 ppm. An initial automated spectral processing step was conducted by HDExaminer followed by a manual inspection of individual peptides for sufficient quality where only one charge state per peptide was kept. Deuterium uptakes were not corrected for back-exchange and are reported as relative. HDX-MS results were statistically validated using an unpaired two-tailed t-test, where significant differences required 0.3 Da, 5% and p-value of <0.05 (n=3)"],"omics_type":["Proteomics"],"labhead":["Roger Lee"],"instrument_platform":[""],"labhead_affiliation":["MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK"],"species":["Homo Sapiens (human)"],"submission_type":["PARTIAL"],"publication":["10.7554/ELIFE.110040.3"],"submitter_mail":["mbourguet@mrc-lmb.cam.ac.uk"],"submitter_affiliation":["MRC Laboratory of Molecular Biology"],"submitter_country":["United Kingdom"],"additional_accession":[]},"is_claimable":false,"name":"A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution","description":"RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII, by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the Saccharomyces cerevisiae sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in S. cerevisiae VPS15 resulted in defective CPY sorting, loss of colocalizsation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.","dates":{"publication":"2026-05-26","submission":"2025-02-27"},"accession":"PXD061277","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:184922","NEWT:6703","NEWT:3555","NEWT:71647","NEWT:2","NEWT:157546","NEWT:474186","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:1496","NEWT:544496","NEWT:2102","NEWT:2042546","NEWT:259447","NEWT:45351","NEWT:445974","NEWT:43179","NEWT:4513","NEWT:180454","NEWT:5722","NEWT:1247","NEWT:6938","NEWT:1129","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:281395","NEWT:10360","NEWT:1211601","NEWT:876138","NEWT:47664","NEWT:317548","NEWT:3654","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:498019","NEWT:1351","NEWT:1438992","NEWT:1352","NEWT:2649997","NEWT:272563","NEWT:1147161","NEWT:224326","NEWT:1333499","NCBITaxon:79857","NEWT:1096976","NEWT:82688","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:5821","NEWT:1589","NEWT:44139","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:28903","NEWT:3880","NEWT:272559","NEWT:28909","NEWT:515849","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:913645","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:143361","NEWT:317447","NEWT:4688","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4442","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NEWT:2164133","NCBITaxon:183674","NEWT:1316931","NEWT:224308","NEWT:167387","NEWT:84645","NEWT:44586","NEWT:626528","NEWT:3347","NEWT:511145","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:5888","NEWT:211586","NEWT:747078","NEWT:1282","NEWT:1283","NEWT:431241","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:2133","NEWT:294381","NEWT:197","NEWT:1390363","NEWT:77133","NEWT:288705","NEWT:29176","NEWT:145481","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:556182","NEWT:1270","NEWT:374990","NEWT:1392","NEWT:498217","NEWT:156471","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:156476","NEWT:286","NEWT:391619","NEWT:360104","NEWT:246196","NEWT:287","NEWT:246197","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:161934","NEWT:1148","NEWT:580240","NEWT:5508","NEWT:294128","NEWT:5507","NEWT:11676","NEWT:55571","NEWT:35500","NEWT:1140","NEWT:100226","NEWT:4530","NEWT:1143","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1390","NEWT:11557","NEWT:1094343","NEWT:1336795","NEWT:644042","NEWT:296543","NEWT:316435","NEWT:42157","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:3712","NEWT:82380","NEWT:1034304","NEWT:105023","NEWT:866628","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:375146","NEWT:9378","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NEWT:8175","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:47946","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:1077286","NEWT:943129","NEWT:243277","NEWT:990119","NEWT:7067","NEWT:2850","NEWT:1111708","NEWT:408172","NEWT:227321","NEWT:408170","NEWT:493760","NEWT:106590","NEWT:260710","NEWT:257313","NEWT:3827","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:930945","NEWT:106592","NEWT:536231","NEWT:46704","NEWT:1436733","NEWT:460519","NEWT:1187947","NEWT:572307","NEWT:1432138","NEWT:269796","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:1194599","NEWT:272844","NEWT:54571","NEWT:9598","NEWT:8030","NEWT:9483","NEWT:1513458","NCBITaxon:40559","NEWT:1639","NEWT:84588","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:759272","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:109757","NEWT:943146","NEWT:2711","NEWT:300852","NEWT:537457","NEWT:1502","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:508771","NEWT:1883446","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:89920","NEWT:29158","NEWT:644223","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:180923","NEWT:108458","NEWT:272623","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:118499","NEWT:169963","NEWT:32644","NEWT:527796","NEWT:225117","NEWT:746128","NEWT:499175","NEWT:109779","NEWT:43665","NEWT:1266464","NEWT:1715989","NCBITaxon:4751","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:3988","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:649908","NEWT:410289","NEWT:373153","NEWT:375451","NEWT:352472","NEWT:357","NEWT:1071661","NEWT:360094","NEWT:470","NEWT:41364","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:1679","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:29491","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:1328426","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NCBITaxon:138","NEWT:40483","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847",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