Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors

Activins are members of the transforming growth factor-beta family of growt h and differentiation factors. In this paper, we report the results of a st ructure-function analysis of activin A. The primary targets for directed mu tagenesis were charged, individual amino acids located in accessible domain s of the protein, concentrating on those that differ from transforming grow th factor-beta 2, the x-ray crystal structure of which is known. Based on t he activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in i nsect cells and purification of these four mutants to homogeneity, they wer e studied in bioassays and in cross-linking experiments involving transfect ed receptor combinations. Mutant D27K has a 2-fold higher specific bio-acti vity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activ ity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and wer e equipotent in bioassays. Our results with the Lys-102 mutants indicate th at the positive charge of amino acid 102 is important for biological activi ty and type II receptor binding of activins.