TGF-β Prodomain Alignments Reveal Unexpected Cysteine Conservation Consistent with Phylogenetic Predictions of Cross-Subfamily Heterodimerization

Evolutionary relationships between prodomains in the TGF-beta family have gone unanalyzed due to a perceived lack of conservation. We developed a novel approach, identified these relationships, and suggest hypotheses for new regulatory mechanisms in TGF-beta signaling. First, a quantitative analysis placed each family member from flies, mice, and nematodes into the Activin, BMP, or TGF-beta subfamily. Second, we defined the prodomain and ligand via the consensus cleavage site. Third, we generated alignments and trees from the prodomain, ligand, and full-length sequences independently for each subfamily. Prodomain alignments revealed that six structural features of 17 are well conserved: three in the straitjacket and three in the arm. Alignments also revealed unexpected cysteine conservation in the LTBP-Association region upstream of the straitjacket and in beta 8 of the bowtie in 14 proteins from all three subfamilies. In prodomain trees, eight clusters across all three subfamilies were present that were not seen in the ligand or full-length trees, suggesting prodomain-mediated cross-subfamily heterodimerization. Consistency between cysteine conservation and prodomain clustering provides support for heterodimerization predictions. Overall, our analysis suggests that cross-subfamily interactions are more common than currently appreciated and our predictions generate numerous testable hypotheses about TGF-beta function and evolution.