IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ fu-2021-1 ecoli Escherichia coli (strain K12) solution nmr solution state nmr solution state nmr solution nmr predetermined predetermined participant predetermined Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins Spike S2 region (1209−1237) was reconstituted in DMPC-DH 6 PC Bicelles that closely mimic a lipid bilayer. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation fu-2021-4 ecoli Escherichia coli (strain K12) comigration in gel comigration in gel electrophoresis comigration in gel weight by comassie molecular weight estimation by coomasie staining weight by comassie Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins Spike S2 region (1209−1237) was reconstituted in DMPC-DH 6 PC Bicelles that closely mimic a lipid bilayer. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation fu-2021-5 in vitro In vitro crosslink cross-linking study crosslink predetermined predetermined participant predetermined Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins Crosslinkers: BS3 (Bissulfosuccinimidyl suberate) and Glutaraldehyde. OG-label (oligomer labeling) method for characterizing the oligomeric state of transmembrane proteins in bicelles: [For extensive further details see PMID: 29551287] A TriNTA-GB1 conjugate is attached to the Spike fragment His-tag. This allows for more efficient cross-linking. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation fu-2021-7 ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column experimental particp experimental participant identification experimental particp Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins His-tagged Spike S2 (1209-1237), as used for OG-label method, was reconstituted in n-Dodecyl-beta-D-Maltopyranoside (DDM) micelles before and after mixing with GB1-TriNTA. The TriNTA-GB1 conjugate is attaches to the Spike fragment His-tag, allowing for more efficient cross-linking. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation fu-2021-2 ecoli Escherichia coli (strain K12) comig non denat gel comigration in non denaturing gel electrophoresis comig non denat gel weight by staining molecular weight estimation by staining weight by staining Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins Spike S2 region (1209−1237) was reconstituted in DMPC-DH 6 PC Bicelles that closely mimic a lipid bilayer. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation fu-2021-3 in vitro In vitro biophysical biophysical predetermined predetermined participant predetermined Fu Q., Chou JJ. Virus - Publications including interactions involving viral proteins Hydrogen-deuterium exchange of Spike S2 TMD in lipid bicelles. Spike S2 (1209-1237) was flash-frozen in liquid nitrogen and lyophilized to remove protonated water. The dried sample was dissolved in D2O. The progress of the H-D exchange was monitored by NMR. james_chou@hms.harvard.edu Journal of the American Chemical Society 2021 imex curation Coronavirus - Interactions investigated in the context of Coronavirus Only protein-protein interactions imex curation spike_sars2 Spike glycoprotein S E2 Peplomer protein 2 protein protein SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 2019-nCoV MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT s-84 4062396 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 13c label 13c label C13 13C C13 13C undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 2h label 2h label deuterium D2 2H2 2H2 deuterium D2 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Table S2 No NMR data was collected for region 1209-1216 as it is unstructured. s-85 4062396 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 13c label 13c label C13 13C C13 13C undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 2h label 2h label deuterium D2 2H2 2H2 deuterium D2 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Table S2, Figure S4,5 No NMR data was collected for region 1209-1216 as it is unstructured. s-88 4062397 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Supp. figure 1C Purified WT Spike S2 1209-1237 (without reconstitution into Bicelles) migrated as a monomer at ~5 kDa s-90 4062397 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Supp. figure 1D Lane 2 s-89 4062398 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false GB1-TriNTA tag tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Supp. figure 1D s-91 4062398 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false GB1-TriNTA tag tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false GB1-TriNTA tag tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Supp. figure 1D Multimers were seen. s-s-14 4062399 N/A 4062402 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Supp. figure 2 Interaction seen in the absence of a TriNTA-GB1 conjugate attached to the Spike fragment His-tag. s-92 4062399 N/A 4062402 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false GB1-TriNTA tag tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false Supp. figure 2 Interaction seen in the presence of a TriNTA-GB1 conjugate attached to the Spike fragment His-tag. s-86 4062400 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P0DTC2:p.Gly1219Tyr mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P0DTC2:p.Ile1221Tyr mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P0DTC2:p.Ala1226Tyr mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P0DTC2:p.Gly1223Tyr mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P0DTC2:p.Ile1225Tyr mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false 3A Purified WT Spike S2 1209-1237 (without reconstitution into Bicelles) migrated as a monomer at ~5 kDa Mutations at L1229Y, and L1233Y are stated to have an effect on trimerisation, but have not been entered here as residues 1229 and 1233 are Methionines, not Leucines. s-87 4062401 N/A 4062402 unspecified role unspecified role neutral component neutral component purified purified Membrane proximal region +TMD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 direct interaction direct interaction false false false 3B Core residues 1225–1229 exhibited the lowest K_ex of all residues.