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 http://mint.bio.uniroma2.it/mint liu-2014-1 Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48-linked ubiquitin chains. in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Liu W., Shang Y., Zeng Y., Liu C., Li Y., Zhai L., Wang P., Lou J., Xu P., Ye Y., Li W. leways@ioz.ac.cn; yihongy@mail.nih.gov leways@ioz.ac.cn,yihongy@mail.nih.gov EMBO J. (0261-4189) 2014 Only protein-protein interactions imex curation liu-2014-3 Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48-linked ubiquitin chains. in vitro In vitro atomic force microsc atomic force microscopy AFM AFM atomic force microsc predetermined predetermined participant predetermined Liu W., Shang Y., Zeng Y., Liu C., Li Y., Zhai L., Wang P., Lou J., Xu P., Ye Y., Li W. leways@ioz.ac.cn; yihongy@mail.nih.gov leways@ioz.ac.cn,yihongy@mail.nih.gov EMBO J. (0261-4189) 2014 Only protein-protein interactions imex curation liu-2014-2 Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48-linked ubiquitin chains. in vitro In vitro bio-layer interferometry bio-layer interferometry bli bli predetermined predetermined participant predetermined Liu W., Shang Y., Zeng Y., Liu C., Li Y., Zhai L., Wang P., Lou J., Xu P., Ye Y., Li W. leways@ioz.ac.cn; yihongy@mail.nih.gov leways@ioz.ac.cn,yihongy@mail.nih.gov EMBO J. (0261-4189) 2014 Only protein-protein interactions imex curation ub2g2_mouse Ubiquitin-conjugating enzyme E2 G2 Ube2g2 Ubiquitin carrier protein G2 Ubiquitin-protein ligase G2 Ubc7 E2 ubiquitin-conjugating enzyme G2 protein protein mouse Mus musculus Mouse MAGTALKRLMAEYKQLTLNPPEGIVAGPMNEENFFEWEALIMGPEDTCFEFGVFPAILSFPLDYPLSPPKMRFTCEMFHPNIYPDGRVCISILHAPGDDPMGYESSAERWSPVQSVEKILLSVVSMLAEPNDESGANVDASKMWRDDREQFYKIAKQIVQKSLGL ube2g2-1 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys48Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f2b f2c f2d f2e High molecular weight crosslinking products were efficiently generated for WT Ube2g2 and the Ube2g2 C75A mutant, but not for the C48A and C89A mutants (Fig 2C–E), suggesting that the crosslinked dimer/oligomers are formed between Cys48 and Cys89 of two neighboring Ube2g2s. Thus, it is likely that in a functional Ube2g2 dimer (see below), Cys48 in one Ube2g2 is in proximity to the catalytic Cys89 of another Ube2g2. This arrangement allows BMH to crosslink multiple Ube2g2 molecules into a higher order oligomer. To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. ube2g2-1 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys89Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f2b f2c f2d f2e High molecular weight crosslinking products were efficiently generated for WT Ube2g2 and the Ube2g2 C75A mutant, but not for the C48A and C89A mutants (Fig 2C–E), suggesting that the crosslinked dimer/oligomers are formed between Cys48 and Cys89 of two neighboring Ube2g2s. Thus, it is likely that in a functional Ube2g2 dimer (see below), Cys48 in one Ube2g2 is in proximity to the catalytic Cys89 of another Ube2g2. This arrangement allows BMH to crosslink multiple Ube2g2 molecules into a higher order oligomer. To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. ube2g2-ube2g2 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys89Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys89Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false f2b f2c f2d f2e High molecular weight crosslinking products were efficiently generated for WT Ube2g2 and the Ube2g2 C75A mutant, but not for the C48A and C89A mutants (Fig 2C–E), suggesting that the crosslinked dimer/oligomers are formed between Cys48 and Cys89 of two neighboring Ube2g2s. Thus, it is likely that in a functional Ube2g2 dimer (see below), Cys48 in one Ube2g2 is in proximity to the catalytic Cys89 of another Ube2g2. This arrangement allows BMH to crosslink multiple Ube2g2 molecules into a higher order oligomer. To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. ube2g2-ube2g2-1 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys48Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys48Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false f2b f2c f2d f2e High molecular weight crosslinking products were efficiently generated for WT Ube2g2 and the Ube2g2 C75A mutant, but not for the C48A and C89A mutants (Fig 2C–E), suggesting that the crosslinked dimer/oligomers are formed between Cys48 and Cys89 of two neighboring Ube2g2s. Thus, it is likely that in a functional Ube2g2 dimer (see below), Cys48 in one Ube2g2 is in proximity to the catalytic Cys89 of another Ube2g2. This arrangement allows BMH to crosslink multiple Ube2g2 molecules into a higher order oligomer. To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. ube2g2-ube2g2-2 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.[Asp98Arg;Asp99Arg;Met101Arg] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f4a f4b The results revealed that several mutants generated fewer crosslinking products when compared to WT Ube2g2 (Fig 4A and B and data not shown). Among these mutants, two [referred to as M3 (D98R/D99R/M101R) and M4 (E129R/R148E/Y152E), respectively] were selected for further characterization because these mutations reduced Ube2g2 dimerization most significantly (Fig 4B). ube2g2-ube2g2-3 1480589 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.[Glu129Arg;Arg148Glu;Tyr152Glu] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f4a f4b The results revealed that several mutants generated fewer crosslinking products when compared to WT Ube2g2 (Fig 4A and B and data not shown). Among these mutants, two [referred to as M3 (D98R/D99R/M101R) and M4 (E129R/R148E/Y152E), respectively] were selected for further characterization because these mutations reduced Ube2g2 dimerization most significantly (Fig 4B). ube2g2-1 1480590 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys48Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f2g f2h In addition, atomic force microscopy (AFM) was used to directly measure the rupture force required to dissociate the E2 dimer for the Ube2g2 variants (Fig 2G). The results showed that the most probable rupture force between two WT Ube2g2 molecules was 24.08 ± 1.19 pN, while between two C48A mutants, it was 17.63 ± 0.56 pN (Fig 2H). ube2g2-ube2g2-4 1480590 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.[Glu129Arg;Arg148Glu;Tyr152Glu] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f4c sf3d-g AFM experiments showed that the most probable rupture forces for the M3 and M4 mutants were significantly reduced comparing with WT Ube2g2 (Fig 4C and supplementary Fig S3D–G). ube2g2-ube2g2-5 1480590 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.[Asp98Arg;Asp99Arg;Met101Arg] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false f4c sf3d-g AFM experiments showed that the most probable rupture forces for the M3 and M4 mutants were significantly reduced comparing with WT Ube2g2 (Fig 4C and supplementary Fig S3D–G). ube2g2-1 1480591 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified P60605:p.Cys48Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 direct interaction direct interaction false false false 1480591 f2f To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. Consistent with our hypothesis, WT Ube2g2 interacted with each other with a KD of around 8 μM, whereas the C48A mutant dimerized with a significantly reduced affinity (Fig 2F, supplementary Fig S3B and C). ube2g2-1 1480591 n/a 1480592 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 2.0 direct interaction direct interaction false false false 1480591 f2f sf3a sf3b sf3c To see if the C48A mutation affected Ube2g2 dimerization, we used Biolayer Interferometry to measure its dimerization activity. Consistent with our hypothesis, WT Ube2g2 interacted with each other with a KD of around 8 μM, whereas the C48A mutant dimerized with a significantly reduced affinity (Fig 2F, supplementary Fig S3B and C).