MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. EMBO Rep. (1469-221X) 2011 imex curation Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. Only protein-protein interactions in vitro In vitro comig non denat gel comigration in non denaturing gel electrophoresis comig non denat gel predetermined predetermined participant predetermined Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. EMBO Rep. (1469-221X) 2011 Only protein-protein interactions imex curation Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. EMBO Rep. (1469-221X) 2011 imex curation Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink weight by comassie molecular weight estimation by coomasie staining weight by comassie Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. EMBO Rep. (1469-221X) 2011 Only protein-protein interactions imex curation Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer. EMBO Rep. (1469-221X) 2011 imex curation Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. Only protein-protein interactions in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Yamasaki M., Sendall TJ., Pearce MC., Whisstock JC., Huntington JA. EMBO Rep. (1469-221X) 2011 Only protein-protein interactions imex curation a1at_human Alpha-1-antitrypsin Alpha-1 protease inhibitor Alpha-1-antiproteinase AAT PI PRO0684 PRO2209 SERPINA1 Serpin A1 protein protein human Homo sapiens Human MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK DIP protein P01009 original sequence version: 186 DIP protein P01009-1 original sequence version: 182 7016555F273B7F16 serpina1-2 3 a1AT 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 3.0 direct interaction direct interaction mint The s5A s6A disulphide variant was created by substituting Cys residues at adjacent positions on s5A and s6A (T339C and S292C). Protein Data Bank: coordinates and structure factors have been deposited with accession code 3T1P. t1 f2b f2c f2d sf3 serpina1-serpina1-2 1 a1AT 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified a1AT 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction homomint mint Incubation of native a1AT either with GndHCl or at elevated temperature resulted in the appearance of smeared bands or interlaced ladders by non-denaturing polyacrylamide gel electrophoresis (PAGE; Fig 1A, left panel), suggesting multiple types of polymers To preclude formation of the s4A/s5A polymer, we engineered a disulphide bond (Cys 292 Cys 339) in a1AT to link strand 5A to strand 6A (supplementary Fig S1D, left panel online). The s5A s6A disulphide variant was resistant to polymerization by GndHCl (supplementary Fig S2 online), but polymers could be formed by incubation at 60 1C (supplementary Fig S2 online, and Fig 1B), and the resulting ladder on native PAGE reacted strongly with the 2C1 antibody (Fig 1B, right panel). Polymerization in vivo was tested by expressing the disulphide variants described above coupled to the polymerigenic Z a1AT mutation in two well-characterized model cell types, Pichia pastoris (Levina et al, 2009) and COS-7 (Miranda et al, 2010). Using a His tag, we were able to purify polymers from the cytosol of P. pastoris that reacted strongly with the 2C1 antibody (supplementary Fig S4 online). Furthermore, purified polymers of Z a1AT with the C-terminal disulphide (Cys216 Cys392) were SDS stable (Fig 3B, lane 3) f1 sf4b serpina1-serpina1-3 1 a1AT 4 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified a1AT 4 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction homomint mint Incubation of native a1AT either with GndHCl or at elevated temperature resulted in the appearance of smeared bands or interlaced ladders by non-denaturing polyacrylamide gel electrophoresis (PAGE; Fig 1A, left panel), suggesting multiple types of polymers To preclude formation of the s4A/s5A polymer, we engineered a disulphide bond (Cys 292 Cys 339) in a1AT to link strand 5A to strand 6A (supplementary Fig S1D, left panel online). The s5A s6A disulphide variant was resistant to polymerization by GndHCl (supplementary Fig S2 online), but polymers could be formed by incubation at 60 1C (supplementary Fig S2 online, and Fig 1B), and the resulting ladder on native PAGE reacted strongly with the 2C1 antibody (Fig 1B, right panel). Polymerization in vivo was tested by expressing the disulphide variants described above coupled to the polymerigenic Z a1AT mutation in two well-characterized model cell types, Pichia pastoris (Levina et al, 2009) and COS-7 (Miranda et al, 2010). Using a His tag, we were able to purify polymers from the cytosol of P. pastoris that reacted strongly with the 2C1 antibody (supplementary Fig S4 online). Furthermore, purified polymers of Z a1AT with the C-terminal disulphide (Cys216 Cys392) were SDS stable (Fig 3B, lane 3) f1 sf4b f3b sf2 serpina1-serpina1-4 2 a1AT 4 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 a1AT 4 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 direct interaction direct interaction homomint mint f3a sf5a