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STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-Å crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their ␤-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells. STAT4 (signal transducer and activator of transcription) proteins mediate the signaling of cytokines and a number of growth factors from the receptors of these extracellular signaling molecules to the cell nucleus. Dependent on the receptor type STATs are specifically phosphorylated by receptor-associated Janus kinases, receptor tyrosine kinases, or cytoplasmic tyrosine kinases (1). The phosphorylated STAT molecules dimerize by reciprocal binding of their SH2 domains to the phosphotyrosine residues. These dimeric STATs translocate into the nucleus, bind to specific DNA sequences, and regulate the transcription of their target genes.Seven mammalian STATs have been identified. Their structural organization is known in molecular detail from several crystal structures. At the N terminus they contain a helical domain, which mediates cooperative binding of STATs to sequential DNA binding sites (2). The structures of the phosphorylated STAT1 and STAT3 core fragments, lacking the N-terminal domain as well as the C-terminal transactivation domain, were solved in complex with DNA (3, 4). They consist of an N-terminal large four-helix bundle, a central IgG-like domain, which constitutes the actual DNA binding domain, a helical, so-called linker domain, and the SH2 domain. The phosphotyrosine residue is located in all mammalian STATs within the 30 amino acids C-terminal to the SH2 domain. In most mammalian STATs the C-terminal part is constituted by a mostly disordered transactivation domain, which mediates the interactions of STATs with other components of the transcription machinery. Three-dimensional information for this domain so far has only been obtained from the complex of a short fragment of the STAT6 transactivation domain with the PAS-B domain of the nuclear receptor coactivator 1 (5).STAT3 homodimers and STAT3-STAT1 complexes have been coimmunoprecipitated from untreated cells (6, 7)...

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