Fig. 4
From: Structural basis of meiotic telomere attachment to the nuclear envelope by MAJIN-TERB2-TERB1
DNA-binding by MAJIN–TERB2. a Surface electrostatic potential of the MAJINCore–TERB2C crystal structure (red, electronegative; blue, electropositive), with SAXS-modelled MAJIN C-terminal extensions in which basic residues are highlighted as blue spheres, and indicating the likely position of basic TERB2 C-terminal ends. Each MAJIN protomer displays a basic surface, which is continuous with the TERB2 C-terminus and basic patches towards the N-terminal end of the MAJIN flexible linkers, providing two extended basic interfaces per MAJIN–TERB2 heterotetramer. b Schematic of the human MAJIN and TERB2 sequences, highlighting the core linker basic patches (BP1 and BP2) and transmembrane helix of MAJIN, and N- and C-terminal domains and C-terminal basic patch (BP) or TERB2. Principal protein constructs are indicated: MAJIN ΔTM (1–233), Core + BP1 (1–147) and Core (1–112); TERB2 C (168–220) and C-Tr (168–207). c EMSA analysing the ability of MAJIN–TERB2 constructs (as indicated) to interact with 0.3 µM (per molecule) linear double-stranded DNA (dsDNA). Gel images are representative of at least three replicate EMSAs. d Quantification of DNA-binding by MAJIN–TERB2 constructs (ΔTM/C, yellow; ΔTM/C-Tr, green; Core/C, black; Core/C F73E Y75E, blue; Core/C-Tr, red; Core/C basic surface mutant, grey) through densitometry of EMSAs performed using 25 nM (per molecule) FAM-dsDNA. Plots and apparent KD values were determined by fitting data to the Hill equation; error bars indicate standard error, n = 3 EMSAs. *Apparent KD was estimated graphically from the concentration at 50% DNA-binding as binding saturation was not achieved. Source data are provided as a Source Data file. e Electron microscopy analysis of MAJINCore-TERB2C and MAJINCore–TERB2C F73E Y75E alone and in complex with plasmid dsDNA. Scale bars, 100 nm. The bottom-right panel shows lack of assembly, through absence of structures of sufficient size for EM visualisation, of MAJINCore–TERB2C F73E Y75E in comparison with wild-type. f Model of the MAJIN–TERB2 complex with dsDNA. A seamless DNA-binding interface is formed on either side of the MAJIN–TERB2 heterotetramer by the MAJIN surface, TERB2 C-terminus and MAJIN linker. A single dsDNA molecule may bind to both surfaces cooperatively through looping around the top of the molecule within the MAJIN linker region.
