The molecular evolutionary history of a winged bean alpha-chymotrypsin inhibitor and modeling of its mutations through structural analyses

A serine protease inhibitor of the Kunitz-STI (soybean trypsin inhibitor) f amily, isolated from the legume seeds of winged bean, was found to inhibit chymotrypsin at a 1:2 stoichiometric ratio. When the structure was determin ed in our laboratory, it was found to form a characteristic beta-trefoil fo ld, which is also seen in other proteins from distant families and sources. The folding organization divides the protein into three approximately equa l subdomains related by a pseudo-threefold axis of symmetry passing paralle l to the barrel axis of the trefoil. Following the now established idea tha t the present-day genes originated from ancestral minigenes through evoluti on, the origin of the proteins having this beta-trefoil organization is scr utinized using its subdomain motif as the search probe. The results, based mainly on structural analyses, indicate the independent existence of such a motif, mimicking the unknown ancestral protein(s) that might have been dis tributed in nature, not only by gene duplication, but also by insertion and permutation in other folds. The understanding led to a hypothesis for the possible origin of the Kunitz-STI family. On the basis of this model of evo lution, structurally hypervariable regions were located on the protein wher e mutations could be designed and a broad range of engineering of the prote in's activity could be conceived.