A rapid conformational rearrangement of STAT1 dimers is required for termination rather than for amplification of interferon-γ signaling

Abstract

Sequence-specific binding of STAT1 (signal transducer and activator of transcription 1) transcription factor to palindromic promoter elements, termed γ-activated sites (GAS), and an extended spatial reorientation between two dimer configurations are key events in the interferon signaling pathway. Although the DNA-binding domain of STAT1 is engaged in both processes, how the conformational change from a parallel to an antiparallel dimer configuration affects cytokine-induced target gene activation is unknown. In order to study the impact of the conformational shift on gene expression, we generated a STAT1 point mutant with a structurally altered architecture of the DNA-binding domain and characterized the resulting mutant (F364A) in cells stimulated with interferon-γ. Here, we report that substituting alanine for phenylalanine at position 364 resulted in reduced affinity to GAS sites and, additionally, a decreased dephosphorylation rate by the inactivating Tc45 phosphatase. The mutant had no defect in cooperative DNA binding and displayed normal kinetics of interferon-γ-induced nuclear accumulation, despite its elevated level of tyrosine phosphorylation. By assessing the transcriptional activity of the mutant, we found a strikingly robust expression of known interferon-γ-driven target genes, indicating that an impaired stability of the antiparallel dimer configuration can compensate for a reduced affinity to GAS sites. However, the mutant followed changes in ligand-induced receptor activation more slowly than the wild-type molecule, as demonstrated by its elevated phospho-STAT1 concentration following addition of the kinase inhibitor staurosporine to interferon-pretreated cells. This finding showed that the DNA-binding mutant F364A had partially lost its ability to terminate signal transmission rapidly. Thus, the coupling of high-affinity GAS binding to a rapid exchange from a parallel to an antiparallel dimer conformation is not necessarily required for optimal signal amplification, but rather allows for a dynamic signal response and ensures high adaptability to changes in signal input.