Phenylalanine 172 in the coiled-coil domain and threonine 385 in the DNA-binding domain (the latter colored in orange and yellow) are part of the interface surface formed by two STAT1 monomers oriented in antiparallel alignment.

<p>(A) Crystal structure of a truncated STAT1 dimer showing the localization of F172 (in magenta) and T385 (in dark blue), including a closer view of the ribbon representation (B), demonstrates the spatial orientation of the functional groups of F172 and T385, which are required for interaction with the partner monomer in the antiparallel dimer conformation. (C) Substitution of alanine for threonine 385 resulted in prolonged and elevated tyrosine phosphorylation of mutant STAT1 in cells stimulated with interferon-γ. Equal numbers of HeLa cells expressing either wild-type (WT) or mutant STAT1, both tagged with green fluorescent protein (GFP), were stimulated with 5 ng/ml of IFNγ for 45 min, before the kinase inhibitor staurosporine (1 µM) was added for the indicated times. Whole cell extracts were assessed for the time course of tyrosine phosphorylation by means of Western blotting using a STAT1-specific phospho-tyrosine antibody followed by a secondary antibody (αp-STAT1, top panel). The same membrane was stripped off bound immunoreactivity and re-probed with the pan-STAT1 antibody C-24 (αSTAT1, bottom panel). The upper arrowhead marks GFP-tagged STAT1 and the lower one indicates endogenous STAT1. (D, E) Gelshift assays confirm the increased DNA-binding activity of the T385A mutant which resulted from enhanced tyrosine phosphorylation. STAT1-negative U3A cells reconstituted with either wild-type or mutant STAT1 were stimulated with IFNγ (5 ng/ml) and subsequently treated with staurosporine for the indicated times, before whole cell lysates were incubated with [³³P]-labeled duplex oligonucleotides containing a GAS site (M67) and loaded onto a native polyacrylamide gel. The asterisk at the right-hand margin of the gel marks a non-specific band and the arrowhead corresponds to recombinant STAT1 bound to M67. (E) Time of DNA binding activity of STAT1-WT and T385A to M67 as quantified from EMSA experiments such as in (D).