A reinterpretation of the dimerization interface of the N-terminal domains of STATs

Abstract

The crystal structures of the N-terminal domain (N-domain) and the core region of the STAT family of transcription factors have been determined previously. STATs can form cooperative higher order structures (tetramers or higher oligomers) while bound to DNA. The crystal packing in the STAT4 N-domain crystal structure, determined at 1.5 A resolution, suggests two alternate organizations of the N-domain dimer. We now present the results of site directed mutagenesis of residues predicted to be involved at each dimer interface. Our results indicate that the dimer interface suggested earlier as being physiologically relevant is, in fact, unlikely to be so. Given the alternative model for the N-domain dimer, the ability of the N-domain to mediate interactions of two STAT dimers on DNA remains unchanged.

Figure 1.

Two alternate organizations of the STAT4 N-domain dimer. (A) The STAT4 N-domain dimer suggested earlier by Vinkemeier et al. (1998). (B) The alternate STAT4 N-domain dimer suggested by crystal packing. Close-up views of dimer interface I (A) and interface II (B) are presented, and the residues involved in dimer formation are indicated. The structures were drawn using Ribbons (Carson 1991), and the PDB coordinates 1BGF for the STAT4 N-domain (Vinkemeier et al. 1998). Starting from the deposited coordinates of the protomer (x,y,z; PDB entry 1BGF), the coordinates for the second molecule in the new dimer (x′,y′,z′) can be generated by x′ = 0.5000*x + 0.8660*y + −0.0000*z + −39.7485; y′ = 0.8660*x + −0.5000*y + 0.0000*z + 68.8534; z′ = 0.0000*x + 0.0000*y + −1.0000*z + 70.5855.

Figure 2.

Analytical ultracentrifugation sedimentation equilibrium data. Each STAT N-domain protein was analyzed at three different concentrations at 25,000 rev/min (see Materials and Methods). Representative results for the wild-type protein and some of the STAT1 N-domain mutant proteins are shown. In each case, the upper panel shows the residual difference between experimental and fitted values by its standard deviation, and the lower panel shows the equilibrium profile. The variances (V) between the fitted and experimental values, and calculated molecular mass (M) in daltons are indicated. The theoretical molecular weight of the STAT1 N-domain monomer is 15,223 Daltons.

Figure 3.

Circular dichroism spectra of STAT1 N-domain proteins. The spectra for wild-type STAT1 N-domain (blue), STAT1 F77A (green), and STAT1 L78A (red) were determined as described in Materials and Methods.