Fig. 2: Structural characterization of DIAPH1–MFN2 interaction.
From: DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress
a Domain structure of the studied proteins. DIAPH1–CFP domains are Rho-BD, Rho GTPase binding domain; DID; DD, dimerization domain; CC, coiled-coil; FH1/2; DAD; and CFP, cyan fluorescent protein, respectively. MFN2 domains are HD1/2, helical domains 1 and 2; and GTase domain. The autoinhibitory intramolecular DID–DAD interaction is indicated. b Binding of the cytosolic MFN2 with DIAPH1-cyan fluorescent protein, CFP, under neutral (blue circles), Kd = 0.7 ± 0.1 nM, and acidic (red circles), Kd = 0.01 ± 0.003 nM, conditions. The binding in the presence of the competitor, DID, is shown by the blue (no binding) and red (Kd = 0.2 ± 0.1 nM) squares under neutral and acidic conditions, correspondingly. Data are presented as mean values ± SEM. c–f. Structural basis of the DID–MFN2 interaction probed by NMR spectroscopy. 1H-15N-HSQC spectra of [U-15N]-DID upon titration with MFN2 and DAD peptide at 305 K. Amide proton–nitrogen cross-peaks from the protein backbone and side chains of 100 µM [U-15N]-DID are broadened upon addition of MFN2 (c, d). Adding DAD peptide (500 µM) restores the cross peaks of DID (e, f). In panel f, red peaks correspond to the NMR spectrum of 100 µM [U-15N]-DID with 100 µM MFN2 and 500 µM DAD, while blue peaks correspond to the NMR spectrum of 50 µM [U-15N]-DID with 200 µM DAD. g A representative high-energy collision MS spectrum was obtained for the DSG cross-linked product between MFN2 and DID at 709.34 m/z. The inlay shows the sequence and composition of cross-linked peptides and an experimental mass of 2128.976 Da, which is in good agreement with the theoretical mass of 2128.992 Da calculated using putative elemental composition. Standard nomenclature was used in labeling charge states and fragment ions. MS data were deposited to ProteomeXchange, https://www.proteomexchange.org/, and jPOST, https://repository.jpostdb.org/, sites with accession numbers PXD045744 and JPST002335, respectively. h Representative ribbon model of the docked MFN2:DIAPH1–DID–DD tetrameric complex. MFN2 and DIAPH1-DID-DD are colored according to the domain structure of panel (a). Source data are provided as a Source Data file.
