PMC:5026484 / 9147-13146 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/5026484","sourcedb":"PMC","sourceid":"5026484","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5026484","text":"10.7554/eLife.18675.004Figure 2. The bMERB domains preferentially interact with Rab8-family members.\n(a) Whereas Rab1 binds to Mical-1, Mical-3 and Mical-cL with low affinity and does not show detectable binding to EHBPs, (b) Rab8 binds with high affinity to all effector domains tested. Additionally, we observed two separate binding sites in the ITC experiments for Rab8 and Mical-1, Mical-3 and EHBP1L1 (the results of the binding fit including the stoichiometry, the KD, the binding enthalpy and the binding entropy are shown within the ITC spectra). (c) Mixing different ratios of Rab8 and the RBDs (1.2:1, 1.6:1 and 2.2:1), the 2:1 stoichiometry of binding was confirmed by aSEC for Rab8:Mical-1 and Rab8:EHBP1L1, whereas a 1:1 stoichiometry was observed for Rab8:Mical-3, Rab8:Mical-cL and Rab8:EHBP1L1 as indicated by a second peak corresponding to free excess Rab8. Note that the second low affinity binding site present in Mical-3 observed via ITC could not be detected via gel filtration.\nDOI: http://dx.doi.org/10.7554/eLife.18675.00410.7554/eLife.18675.005Figure 2—figure supplement 1. Interaction of Rab proteins with the RBD of Mical-1, Mical-3, Mical-cL, EHBP1 and EHBP1L1.\nThe Rab protein (130 µM, black) preparatively loaded with the non-hydrolyzable GTP-analogue GppNHp, the different bMERB domains (112.5 µM, red) and the mixture of both (130 µM Rab + 112.5 µM RBD) were subjected to analytical size exclusion chromatography (aSEC) and tested for complex formation between the RBDs (from left to right: Mical-1, Mical-3, Mical-cL, EHBP1, EHBP1L1) and the Rabs ((a) – Rab1, (b) – Rab8, (c) – Rab10, (d) – Rab13, (e) – Rab15). Note the missing (Rab1:EHBP1 and Rab1:EHBP1L1) or incomplete (Rab1:Mical-3 and Rab1:Mical-cL) complex formation in (a) indicating low-affinity binding.\nDOI: http://dx.doi.org/10.7554/eLife.18675.005\n10.7554/eLife.18675.006Figure 2—figure supplement 2. Interaction of Rab1, Rab35, Rab8, Rab10, Rab13 and Rab15 with Mical-cL.\n(a) The ITC data nicely show the relatively weak affinity of Rab1 towards Mical-cL (KD = 5.2 µM) compared to Rab8 (KD = 233 nM), Rab10 (KD = 787 nM), Rab13 (KD = 93 nM) and Rab15 (KD = 33 nM). Rab35 (sometimes also referred to as Rab1c) has an intermediate affinity for Mical-cL (KD = 1.8 µM). Since preparative loading of Rab35 with GppNHp (see below) was not successful, the concentration of the Rab35 protein fraction in the active state was deduced from HPLC analysis (see below) and the ITC data was corrected accordingly. (b) Comparison of the nucleotide status of Rab8 and Rab35 by HPLC analysis (black trace – absorption at 254 nm, red trace – absorption at 280 nm). The lower lane shows the trace of a nucleotide mix consisting of guanosine, GMP, GDP, GppNHp and GTP and a small impurity of GppNH2 as a reference, the retention times (in minutes) for each nucleotide are shown above the peak and in the table on the right. Whereas Rab8 could be preparatively loaded with GppNHp and analysis showed approximately 94% bound GppNHp, Rab35 was only 82% GppNHp-bound and the trace shows a major impurity of GppNH2 (18%). For this reason, the Rab35 protein concentration used in the ITC data in (a) was corrected for this.\nDOI: http://dx.doi.org/10.7554/eLife.18675.006\n10.7554/eLife.18675.007Figure 2—figure supplement 3. Formation of a ternary complex between Rab8, Rab13 and the RBD of Mical1 or EHBP1L1.\nRab8:GppNHp (130 µM, black), Rab13:GppNHp (130 µM, green) the different bMERB domains (112.5 µM, red) and the mixture of both (130 µM Rab + 112.5 µM RBD; dark blue for Rab8 and magenta for Rab13 in complex with the RBD) were subjected to analytical size exclusion chromatography (aSEC) and tested for complex formation between the Rabs and the RBDs. Additionally, the proteins were mixed in a ratio of 130 µM Rab8, 130 µM Rab13 and 112.5 µM RBD (light blue), clearly indicating that a 1:1:1 complex can be formed with 2 different Rab proteins bound to the respective RBDs.\nDOI: http://dx.doi.org/10.7554/eLife.18675.007","divisions":[{"label":"Title","span":{"begin":33,"end":100}},{"label":"Figure caption","span":{"begin":1046,"end":1843}},{"label":"Title","span":{"begin":1099,"end":1189}},{"label":"Figure caption","span":{"begin":1844,"end":3241}},{"label":"Title","span":{"begin":1897,"end":1968}},{"label":"Title","span":{"begin":3295,"end":3379}}],"tracks":[]}