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 cell-mediated immunity. Upon activation, dimers of STAT4 bind cooperatively to tandem interferon-gamma activation sequences (GAS elements) near the interferon-gamma gene and stimulate its transcription. The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-gamma transcription in response to interleukin-12. We have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to homogeneity for structural studies. The circular dichroism spectrum of hSTAT4(1-124) indicates that it has a well ordered conformation in solution. The translational diffusion constant of hSTAT4(1-124) was determined by nuclear magnetic resonance methods and found to be consistent with that of a dimer. The rotational correlation time (tauc) of hSTAT4(1-124) was estimated from 15N relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein. These results, together with the number of signals observed in the two-dimensional 1H-15N heteronuclear single quantum coherence spectrum of uniformly 15N-labeled protein, indicate that hSTAT4(1-124) forms a stable, symmetric homodimer in solution. Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-terminal domains of adjacent dimers bound to DNA.
Baden
H A
HA
Protein Engineering Group, ABL Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.
Sarma
S P
SP
Kapust
R B
RB
Byrd
R A
RA
Waugh
D S
DS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D050797:STAT4 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050797:STAT4 Transcription Factor; C494090:STAT4 protein, human; D015534:Trans-Activators