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 Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization. Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization. FEBS J. (1742-464X) 2011 imex curation White GF., Singleton C., Todd JD., Cheesman MR., Johnston AW., Le Brun NE. Only protein-protein interactions in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined White GF., Singleton C., Todd JD., Cheesman MR., Johnston AW., Le Brun NE. FEBS J. (1742-464X) 2011 Only protein-protein interactions imex curation Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization. Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization. FEBS J. (1742-464X) 2011 imex curation White GF., Singleton C., Todd JD., Cheesman MR., Johnston AW., Le Brun NE. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined White GF., Singleton C., Todd JD., Cheesman MR., Johnston AW., Le Brun NE. FEBS J. (1742-464X) 2011 Only protein-protein interactions imex curation q8klu1_rhilv Ferric uptake regulation protein irr RLV_7125 fur HB774_20080 E0H54_04310 protein protein rhilv Rhizobium leguminosarum bv. viciae MTGALPIAIEVRLRGAGLRPTRQRVALGDLLFAKGDRHLTVEELHEEAVAAGVPVSLATVYNTLHQFTEAGLIRVLAVESAKTYFDTNVSDHHHFFVEGENEVLDIPVSNLTIANLPEPPEGMEIAHVDVVIRLRAKQG mint BE8A8D787EE19052 irr-1 2 irr 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 2.0 direct interaction direct interaction mint To analyse the association state of IrrRl, analytical gel filtration experiments were performed. For the as-isolated, heme-free protein, two distinct peaks were detected in the chromatograph (Figure 3A). Thus, the heme-free protein exists as an equilibrium mixture of two principal association states, which must be in slow exchange, such that they can be separated by gel filtration. Calibration of the column suggested that the lower molecular mass species corresponds to a dimeric form, consistent with our previous proposal that heme bound at the HxH motif of IrrRl is EPR-silent as a result of magnetic coupling between two closely located HxH-bound hemes. The higher molecular mass species corresponded to a much larger, oligomeric form, but this could not be precisely defined through gel filtration alone. Analysis of elution data at different wavelengths confirmed that the observed profile changes were due to a change in the distribution between higher and lower molecular mass species and not due to preferential binding of heme to the larger species (Supplementary Figure S4). f3a f3d f4 sf4 irr-1 2 irr 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 6.0 direct interaction direct interaction f3d mint To examine the role (if any) of the HxH motif in the heme-dependent oligomerization, the association state of the H93/94/95A mutant IrrRl was also investigated. The gel filtration profile (Figure 3D) was very similar to that of the wild-type protein, and the addition of heme to the mutant polypeptide caused a similar shift in equilibrium towards the larger (hexameric) species. irr-1 1 irr 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 6.0 direct interaction direct interaction f3c mint To determine the mass of the oligomeric species more precisely, analytical ultracentrifugation experiments were run for heme-IrrRl at five different concentrations, ranging from 5 19 μM, at 15,000 and at 17,000 rpm. Figure 3C shows data for one representative concentration of heme-IrrRl (10 μM). Fitting of the data, at both speeds and for all concentrations, to a single component model gave a mass of 96.6 ±8 kDa for the heme-bound IrrRl sample (residuals for the fits are shown inset). The data thus indicate that the higher molecular weight species of IrrRl corresponds to a hexamer.