Fig. 2: Experimental design and determination of the domain architecture of L-TGF-β1/GARP. | Nature Communications

Fig. 2: Experimental design and determination of the domain architecture of L-TGF-β1/GARP.

From: Latent-TGF-β has a domain swapped architecture

Fig. 2

a Ribbon diagrams of two possible architectures of L-TGF-β1 predicted by AlphaFold, in which the straitjacket domain of each monomer is connected ipsilaterally (left) or contralaterally (right) with its arm domain. Domain arrangement is illustrated under the ribbon diagrams. Color scheme in the ribbon diagram and domain arrangements are the same. b Plasmid constructs of L-TGF-β1_R249A_RGE_lasso1 (left), L-TGF-β1_R249A_RGD_lasso3 (middle), and GARP (right). c Anticipated expression products and their proportions from the 1:1:1 transfection of Expi293 cells. Nomenclature of the expected products is shown (heterodimer DL3-EL1 = GARP/L-TGF-β1_RGD_lasso3/L-TGF-β1_RGE_lasso1; homodimer DL3-DL3 = GARP/L-TGF-β1_RGD_lasso3/L-TGF-β1_RGD_lasso3; homodimer EL1-EL1 = GARP/L-TGF-β1_RGE_lasso1/L-TGF-β1_RGE_lasso1). Ribbon diagrams of L-TGF-β1 in ipsilateral (upper) or contralateral (bottom) architectures are predicted by AlphaFold. d SEC profile (middle) of purified mutant L-TGF-β1/GARP incubated with αvβ8 ectodomain (left). The shaded peak contains L-TGF-β1/GARP bound with a single αvβ8 with two possible architectures (bottom). e Incubation of monoclonal antibody clone 28G11 with the SEC-purified complex. f ELISA confirms that 28G11 only binds lasso1 but not lasso3. Data is presented as mean of absorbance450 (ABS450) ±SEM (N = 4 biologic replicates), P-values were generated by two way ANOVA with repeated measures followed by Šídák’s multiple comparisons test. Source data are provided as a Source Data file. g The only possible models resulting from binding of 28G11 to mutant heterodimeric L-TGF-β1(DL3-EL1)/GARP in ipsilateral (left) or contralateral (right) architectures. The flexible regions of bound 28G11 IgG are indicated by cartoons. h Representative 2D class averages and 3D reconstruction with atomic models of a generic Fab and contralateral L-TGF-β1/GARP docked. GARP is partially seen in 2D class averages, but not in the 3D reconstruction. Part of mature TGF-β1 and remaining of IgG are illustrated but not seen in the density map.

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