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 Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by TCA cycle metabolites. Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by TCA cycle metabolites. FEBS J. (1742-464X) 2013 imex curation Bartholomae M., Meyer F.M., Commichau F.M., Burkovski A., Hillen W., Seidel G. gseidel@biologie.uni-erlangen.de 18-Feb-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Maike Bartholomae1, Frederik M. Meyer2, Fabian M. Commichau2, Andreas Burkovski1, Wolfgang Hillen1†, Gerald Seidel gseidel@biologie.uni-erlangen.de Accepted 2013-DEC-09 by LUANA 2013 imex curation Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by TCA cycle metabolites. Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by TCA cycle metabolites. FEBS J. (1742-464X) 2013 imex curation Bartholomae M., Meyer F.M., Commichau F.M., Burkovski A., Hillen W., Seidel G. gseidel@biologie.uni-erlangen.de 18-Feb-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Maike Bartholomae1, Frederik M. Meyer2, Fabian M. Commichau2, Andreas Burkovski1, Wolfgang Hillen1†, Gerald Seidel gseidel@biologie.uni-erlangen.de Accepted 2013-DEC-09 by LUANA 2013 imex curation mdh_bacsu Malate dehydrogenase mdh Vegetative protein 69 citH BSU29120 protein protein bacsu Bacillus subtilis MGNTRKKVSVIGAGFTGATTAFLIAQKELADVVLVDIPQLENPTKGKALDMLEASPVQGFDAKITGTSNYEDTAGSDIVVITAGIARKPGMSRDDLVSTNEKIMRSVTQEIVKYSPDSIIVVLTNPVDAMTYAVYKESGFPKERVIGQSGVLDTARFRTFVAEELNLSVKDVTGFVLGGHGDDMVPLVRYSYAGGIPLETLIPKERIDAIVERTRKGGGEIVNLLGNGSAYYAPAASLTEMVEAILKDQRRVLPTIAYLEGEYGYEGIYLGVPTIVGGNGLEQIIELELTDYERAQLNKSVESVKNVMKVLS mint 656BA5BF5AA2D0CD idh_bacsu Isocitrate dehydrogenase [NADP] icd citC BSU29130 Oxalosuccinate decarboxylase NADP(+)-specific ICDH IDP protein protein bacsu Bacillus subtilis MAQGEKITVSNGVLNVPNNPIIPFIEGDGTGPDIWNAASKVLEAAVEKAYKGEKKITWKEVYAGEKAYNKTGEWLPAETLDVIREYFIAIKGPLTTPVGGGIRSLNVALRQELDLFVCLRPVRYFTGVPSPVKRPEDTDMVIFRENTEDIYAGIEYAKGSEEVQKLISFLQNELNVNKIRFPETSGIGIKPVSEEGTSRLVRAAIDYAIEHGRKSVTLVHKGNIMKFTEGAFKNWGYELAEKEYGDKVFTWAQYDRIAEEQGKDAANKAQSEAEAAGKIIIKDSIADIFLQQILTRPNEFDVVATMNLNGDYISDALAAQVGGIGIAPGANINYETGHAIFEATHGTAPKYAGLDKVNPSSVILSGVLLLEHLGWNEAADLVIKSMEKTIASKVVTYDFARLMDGATEVKCSEFGEELIKNMD mint CC69E694EB66D0D8 mdh-icd 2 Mdh 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Idh 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f1 f2 the interaction of Mdh and Icd was characterized in the presence of their substrates and products as well. Therefore, isocitrate, α-ketoglutarate, malate, and oxaloacetate, as well as the cofactors Mg2+, NADP+, and NADPH for Icd, and NAD+ and NADH for Mdh were added alone, in double and triple combinations. The concentrations of substrates and cofactors were adjusted to the respective intracellular concentrations which were determined for B. subtilis and E. coli in the exponential growth phase and at high availability of carbon and inorganic nitrogen sources, respectively [20-23]. The sensorgrams of these SPR analyses are shown in Figure 2. The single substances and their double combinations or the triple combinations of Mg2+, α- ketoglutarate and NADPH, of Mg2+, malate and NAD+ and of Mg2+, oxaloacetate and NADH did not show significant changes of the sensorgram for 20 μM Icd. Only the combination of substrate and cofactors of Icd, isocitrate, NADP+ and Mg2+, stimulated the interaction of Mdh and Icd to a high level (Figure 2). Thus, the stimulatory effect of the triple combination of the substrates and cofactors of Icd is highly specific. icd-mdh 1 Mdh 3 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Idh 4 predetermined predetermined participant predetermined western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell strep tag strep tag strep tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f3 As shown in Figure 3, FLAG-Mdh stability was not affected by formaldehyde treatment because equal amounts of Mdh were detected in the crude extracts before purification of Strep-Icd or Strep-IcdS104P. Small amounts of the FLAG-tagged Mdh were detected in the fractions from non-cross-linked cultures when the Strep-tagged wildtype and the Icd mutant variant served as the bait proteins. In contrast, the amount of co-purified FLAG-Mdh was increased when both wildtype Strep-Icd and Strep-IcdS104P were purified from the cross-linked cells. Thus, mutant and wildtype Icd show the same interaction pattern with Mdh in vivo and we concluded that the mutation of serine 104 to proline in Icd did not affect its interaction with Mdh. mdh-icd-1 2 Mdh 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Idh 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified P39126:p.Ser104Pro mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain S P Wed May 02 15:11:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser104pro' Tue Jun 30 18:19:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser104Pro' his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f4 the interaction of Mdh and Icd was characterized in the presence of their substrates and products as well. Therefore, isocitrate, α-ketoglutarate, malate, and oxaloacetate, as well as the cofactors Mg2+, NADP+, and NADPH for Icd, and NAD+ and NADH for Mdh were added alone, in double and triple combinations. The concentrations of substrates and cofactors were adjusted to the respective intracellular concentrations which were determined for B. subtilis and E. coli in the exponential growth phase and at high availability of carbon and inorganic nitrogen sources, respectively [20-23]. The sensorgrams of these SPR analyses are shown in Figure 2. The single substances and their double combinations or the triple combinations of Mg2+, α- ketoglutarate and NADPH, of Mg2+, malate and NAD+ and of Mg2+, oxaloacetate and NADH did not show significant changes of the sensorgram for 20 μM Icd. Only the combination of substrate and cofactors of Icd, isocitrate, NADP+ and Mg2+, stimulated the interaction of Mdh and Icd to a high level (Figure 2). Thus, the stimulatory effect of the triple combination of the substrates and cofactors of Icd is highly specific. The interaction of IcdS104P with Mdh was analysed by SPR in the presence or the absence of substrates, cofactors and products, and their double or triple combinations as described before. Again only the triple combination of 1 mM isocitrate, 0.5 mM NADP+ and 5 mM MgCl2 stimulated binding of the IcdS104P mutant to Mdh and, therefore, this mutant was a promising candidate for a quantitative analysis of the interaction. Interestingly, the sensorgrams of the analysis of IcdS104P with Mdh without substrates showed a stronger interaction than wildtype Icd (Figure 4). The kinetic analysis revealed an association rate constant (ka) of 2.0±0.2•102 M-1 s-1, a dissociation rate constant (kd) of 1.0±0.1•10-3 s-1 and an equilibrium constant (KD) of 5.0±0.1 μM (χ²=1.8-4.8). For the kinetic analysis of the IcdS104P-Mdh interaction in the presence of substrates and cofactors, 0.5 to 20 μM IcdS104P were injected with 1 mM isocitrate, 0.5 mM NADP+ and 5 mM MgCl2 on a Mdh-coated CM5 chip. The kinetic analysis revealed an association rate constant (ka) of 1.7±0.7•103 M-1 s-1, a dissociation rate constant (kd) of 2.6±0.6•10-4 s-1 and an equilibrium constant (KD) of 150±1 nM (χ²=2.3-3.0). The comparison of the quantitative data shows a 33-fold stimulation of the interaction of IcdS104P, and the KD values are in a reasonable range for protein-protein interactions.