THE AMINO-TERMINAL DOMAIN OF HUMAN STAT4 - OVERPRODUCTION, PURIFICATION, AND BIOPHYSICAL CHARACTERIZATION

The multifunctional signal transducer and activator of transcription ( STAT) proteins relay signals from the cell membrane to the nucleus in response to cytokines and growth factors. STAT4 becomes activated when cells are treated with interleukin-12, a key cytokine regulator of ce ll-mediated immunity. Upon activation, dimers of STAT4 bind cooperativ ely to tandem interferon-gamma activation sequences (GAS elements) nea r the interferon-gamma gene and stimulate its transcription. The amino -terminal domain of STAT4 (STAT4(1-124)) is required for cooperative b inding interactions between STAT4 dimers and activation of interferon- gamma transcription in response to interleukin-12. me have overproduce d this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and p urified it to homogeneity for structural studies. The circular dichroi sm spectrum of hSTAT4(1-124) indicates that it has a well ordered conf ormation in solution. The translational diffusion constant of hSTAT4(1 -124) was determined by nuclear magnetic resonance methods and found t o be consistent with that of a dimer. The rotational correlation time (tau(c)) of hSTAT4(1-124) was estimated from N-15 relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein. These resu lts, together with the number of signals observed in the two-dimension al H-1-N-15 heteronuclear single quantum coherence spectrum of uniform ly N-15-labeled protein, indicate that hSTAT4(1-124) forms a stable, s ymmetric homodimer in solution. Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-ter minal domains of adjacent dimers bound to DNA.