Evolutionary trace analysis of TGF-beta and related growth factors: implications for site-directed mutagenesis.

The TGF-beta family of growth factors contains a large number of homologous proteins, grouped in several subfamilies on the basis of sequence identity. These subgroups can be combined into three broader groups of related cytokines, with marked specificities for their cellular receptors: the TGF-betas, the activins and the BMPs/GDFs. Although structural information is available for some members of the TGF-beta family, very little is known about the way in which these growth factors interact with the extra-cellular domains of their multiple cell surface receptors or with the specific protein inhibitors thought to modulate their activity. In this paper, we use the evolutionary trace method [Lichtarge et al. (1996) J. Mol. Biol., 257, 342-358] to locate two functional patches on the surface of TGF-beta-like growth factors. The first of these is centred on a conserved proline (P(36) in TGF-betas 1-3) and contains two amino acids which could account for the receptor specificity of TGF-betas (H(34) and E(35)). The second patch is located on the other side of the growth factor protomer and surrounds a hydrophobic cavity, large enough to accommodate the side chain of an aromatic residue. In addition to two conserved tryptophans at positions 30 and 32, the main protagonists in this potential binding interface are found at positions 31, 92, 93 and 98. Several mutagenesis studies have highlighted the importance of the C-terminal region of the growth factor molecule in TGF-betas and of residues in activin A equivalent to positions 31 and 94 of the TGF-betas for the binding of type II receptors to these ligands. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.