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/ li-2004e-1 Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes. ecoli Escherichia coli (strain K12) toxcat tox-r dimerization assay toxcat elisa enzyme linked immunosorbent assay ELISA ELISA elisa Li R., Gorelik R., Nanda V., Law PB., Lear JD., DeGrado WF., Bennett JS. TOXCAT: The chimeric protein consiting of an amino-terminal ToxR' DNA binding domain , a transmembrain domain of interest and a MBP tag. The dimerization was tested by MalE complementation and quantified using CAT (Chloramphenicol Acetyltransferase) ELISA. The orientation in the E. coli membrane of each ToxR-TM-MBP chimeric proteins were checked by proteinase digestion of spheroblasts. fig 2, fig 3, fig 4, fig 6 bennetts@mail.med.upenn.edu J. Biol. Chem. (0021-9258) 2004 IMEx Only protein-protein interactions imex curation imex curation li-2004e-2 Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes. in vitro In vitro 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 Li R., Gorelik R., Nanda V., Law PB., Lear JD., DeGrado WF., Bennett JS. TOXCAT: The chimeric protein consiting of an amino-terminal ToxR' DNA binding domain , a transmembrain domain of interest and a MBP tag. The dimerization was tested by MalE complementation and quantified using CAT (Chloramphenicol Acetyltransferase) ELISA. The orientation in the E. coli membrane of each ToxR-TM-MBP chimeric proteins were checked by proteinase digestion of spheroblasts. fig 2, fig 3, fig 4, fig 6 bennetts@mail.med.upenn.edu J. Biol. Chem. (0021-9258) 2004 IMEx Only protein-protein interactions imex curation imex curation li-2004e-3 Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes. in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined Li R., Gorelik R., Nanda V., Law PB., Lear JD., DeGrado WF., Bennett JS. TOXCAT: The chimeric protein consiting of an amino-terminal ToxR' DNA binding domain , a transmembrain domain of interest and a MBP tag. The dimerization was tested by MalE complementation and quantified using CAT (Chloramphenicol Acetyltransferase) ELISA. The orientation in the E. coli membrane of each ToxR-TM-MBP chimeric proteins were checked by proteinase digestion of spheroblasts. fig 2, fig 3, fig 4, fig 6 bennetts@mail.med.upenn.edu J. Biol. Chem. (0021-9258) 2004 IMEx Only protein-protein interactions imex curation imex curation glpa_human Glycophorin-A PAS-2 Sialoglycoprotein alpha MN sialoglycoprotein GYPA GPA protein protein human Homo sapiens Human MYGKIIFVLLLSEIVSISASSTTGVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ ita2b_human Integrin alpha-IIb Platelet membrane glycoprotein IIb GPalpha IIb ITGA2B GP2B ITGAB protein protein human Homo sapiens Human MARALCPLQALWLLEWVLLLLGPCAAPPAWALNLDPVQLTFYAGPNGSQFGFSLDFHKDSHGRVAIVVGAPRTLGPSQEETGGVFLCPWRAEGGQCPSLLFDLRDETRNVGSQTLQTFKARQGLGASVVSWSDVIVACAPWQHWNVLEKTEEAEKTPVGSCFLAQPESGRRAEYSPCRGNTLSRIYVENDFSWDKRYCEAGFSSVVTQAGELVLGAPGGYYFLGLLAQAPVADIFSSYRPGILLWHVSSQSLSFDSSNPEYFDGYWGYSVAVGEFDGDLNTTEYVVGAPTWSWTLGAVEILDSYYQRLHRLRGEQMASYFGHSVAVTDVNGDGRHDLLVGAPLYMESRADRKLAEVGRVYLFLQPRGPHALGAPSLLLTGTQLYGRFGSAIAPLGDLDRDGYNDIAVAAPYGGPSGRGQVLVFLGQSEGLRSRPSQVLDSPFPTGSAFGFSLRGAVDIDDNGYPDLIVGAYGANQVAVYRAQPVVKASVQLLVQDSLNPAVKSCVLPQTKTPVSCFNIQMCVGATGHNIPQKLSLNAELQLDRQKPRQGRRVLLLGSQQAGTTLNLDLGGKHSPICHTTMAFLRDEADFRDKLSPIVLSLNVSLPPTEAGMAPAVVLHGDTHVQEQTRIVLDCGEDDVCVPQLQLTASVTGSPLLVGADNVLELQMDAANEGEGAYEAELAVHLPQGAHYMRALSNVEGFERLICNQKKENETRVVLCELGNPMKKNAQIGIAMLVSVGNLEEAGESVSFQLQIRSKNSQNPNSKIVLLDVPVRAEAQVELRGNSFPASLVVAAEEGEREQNSLDSWGPKVEHTYELHNNGPGTVNGLHLSIHLPGQSQPSDLLYILDIQPQGGLQCFPQPPVNPLKVDWGLPIPSPSPIHPAHHKRDRRQIFLPEPEQPSRLQDPVLVSCDSAPCTVVQCDLQEMARGQRAMVTVLAFLWLPSLYQRPLDQFVLQSHAWFNVSSLPYAVPPLSLPRGEAQVWTQLLRALEERAIPIWWVLVGVLGGLLLLTILVLAMWKVGFFKRNRPPLEEDDEEGE gypa-gypa-1 TOXCAT: Dimerization of glycophorin A TM region 233073 N/A 233076 unspecified role unspecified role bait bait transmembrane region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P02724:p.Gly102Ile mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false mbp tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false toxr tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 233076 unspecified role unspecified role prey prey transmembrane region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false gly102ile sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false mbp tag tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false toxr tag tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false 233085 233080 physical association physical association false false false Fig. 3 itga2b-itga2b-1 TOXCAT: Dimerization of alpha II b integrin TM region 233073 N/A 233077 unspecified role unspecified role bait bait P08514:p.Gly1003Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1006Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Met1018Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false transmembrane region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1000Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1003Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1006Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1004Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Thr1012Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Ala1017Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1001Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1002Ile mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1003Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1014Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1015Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false leu1011val mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Thr1012Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Thr1012Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Ala1017Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1002Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Val1002Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1011Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Ile1013Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1003Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false N/A 233077 unspecified role unspecified role prey prey transmembrane region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 233119 233094 physical association physical association false false false Fig. 5 The orientation of the alpha II b integrin transmembrane helix in the bacterial membrane of E. coli is opposite to its orientation in eukaryotic membranes. The residues preceding the the critical glycines GXXXG are important for dimerization whereas the succeeding residues were found to be substantially more mutable. itga2b-itga2b-2 SDS-PAGE: oligomerization of alpha II b integrin 233074 N/A 233077 unspecified role unspecified role neutral component neutral component tm-cyto region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1011Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 233077 unspecified role unspecified role neutral component neutral component tm-cyto region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1011Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) 233126 233122 direct interaction direct interaction false false false Fig. 7A The oligomerization of alpha II b integrin fragment containing the transmembrane region and the cytoplasmic region is only detected as a dimer on SDS-PAGE. The mutation Leu1011Ala increases the oligomerization and a faint band of trimer is also detected. itga2b-itga2b-3 Ultracentrifugation: oligomerization of alpha II b integrin 233075 N/A 233077 unspecified role unspecified role neutral component neutral component P08514:p.Leu1011Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1003Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false tm-cyto region 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) N/A 233077 unspecified role unspecified role neutral component neutral component tm-cyto region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P08514:p.Leu1011Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1003Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P08514:p.Gly1007Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false The data of oligomerization of alpha II b integrin fragment containing the transmembrane region and the cytoplasmic region is best fitted with a monomer-dimer equilibrium. The mutant Leu1011Ala data could also be fitted with a monomere-dimer-trimer equilibrium.