Fig. 1. Predicted structure and dynamics characteristics of the wild-type Mef2D and variant sequences.

a Mef2D is extensively disordered and predicted to form liquid-liquid phase separated condensates. The Mef2D structure predicted by Alphafold⁵⁷ indicates a small structured domain involving the N-terminal ~100 residues and most of the transactivation domain (TAD) contains is disordered. The regions promoting formation of liquid-like droplets by the FuzDrop method⁵⁸ are marked by blue. The β-domain (magenta) appears as an ordered motif within the disordered transactivation region. FuzDrop predictions²⁶ shown on the right panels indicate high droplet-promoting probability (p_DP) in particular for regions 155-268 residues and 341-520 residues, which are predicted to spontaneously form liquid-liquid phase separated condensates. The β-domain (magenta) and its flanking regions (cyan) are predicted to serve as ordered interaction motifs within the condensate (see also Supplementary Fig. 1). In addition, the β-domain region is capable of sampling a multiplicity of binding modes (MBM), indicating its sensitivity to the cellular context. b Sequences of the designed Mef2D variants. The β-domain and its flanking regions are shown for the wild-type (wt) Mef2D (UniProt code: Q14814; https://legacy.uniprot.org/uniprot/Q14814; 265-301 residues), var1 and var2 with similar β-domain dynamics (gray), var3 and var4 with mobile β-domains (green), var5-var8 with rigid β-domains (red) as compared to wild-type Mef2D. The sequence of the β-domain is magenta, mutated residues (orange) are highlighted. c Predicted β-domain disorder of Mef2D variants. Structural disorder in the unbound state of Mef2D were computed for the full protein sequence using the Espritz method³⁰ as embedded in the FuzPred program²⁵ and the p_D values were averaged for residues 286-292. The var3 and var4 variants (green) are above the threshold between disorder and order (p_D ≥ 0.3085³⁰). var1 (gray) has similar, var2 (gray) has slightly more mobile β-domain than the wild-type Mef2D (black). var5-var8 variants (red) are predicted to have more rigid β-domain than the wild-type. d Droplet landscape of the Mef2D variants. The droplet landscape shows the droplet probability (p_DP) as a function of the multiplicity of binding modes (MBM)^31,35. The assemblies below the diagonal are likely more solid-like, those above the diagonal are more liquid like³³. High MBM values indicate an increased likelihood to change between liquid-like and solid-like forms, for example in case of var8. More mobile β-domain variants (var3, var4, green diamonds) exhibit increased probability to form droplets (higher p_DP), whereas more rigid β-domain variants (var5-var8, red triangles) more likely form solid-like states depending on the cellular conditions (high MBM).