An avidity-driven mechanism of extracellular BMP regulation by Twisted gastrulation

Bone Morphogenetic Protein (BMP) signalling is tightly regulated extracellularly by specific protein-protein interactions. During dorsoventral patterning of vertebrate and invertebrate embryos, the conserved regulator Twisted gastrulation (Tsg) precisely modulates BMP signalling by binding Chordin/Short gastrulation (Sog) to promote formation of the inhibitory Tsg-Sog/Chordin-BMP ternary complex. Here we elucidate the mechanism by which Tsg interacts with Sog/Chordin to modulate BMP signalling extracellularly. Using AlphaFold predictions, we identify a Chordin binding epitope in the Tsg C-terminal domain, which we validate using in vitro binding studies with targeted point mutants. Introduction of the equivalent point mutation into Drosophila Tsg, to disrupt Tsg-Sog interaction, results in an unexpectedly mild perturbation to embryonic dorsoventral patterning in vivo, in the form of a shallower BMP gradient. Using binding assays, we provide a molecular explanation for this mild phenotype by showing that the BMP ligand can partially rescue ternary complex formation when the Tsg-Sog interaction is disrupted. Additionally, we show that an evolutionary divergent Tsg C-terminal extension is essential for full Tsg function in Drosophila embryos. Based on these findings we propose that Tsg promotes formation of a Tsg-Sog/Chordin-BMP complex by an avidity-driven mechanism, which will be relevant to a broad range of developmental contexts.