W0181

Structure Solution of the N-Terminal Domain of the Doublecortin-Like Kinase From a Single Se. M. H. Kim, U. Derewenda, Y. Devedjiev and Z. S. Derewenda, Dept. of Mol. Physiology and Biological Physics, Univ. of Virginia, Charlottesville VA, 22908

The crystal structure of the N-terminal DCX domain (N-DCLK) of the doublecortin-like kinase (DCLK) was solved using a single Se atom. The N-DCLK (residues 49-154) was subcloned into pGSTUni1 vector, expressed and purified by standard methods. Since the domain does not contain any methionines, a single site mutant L120M was prepared. The SeMet crystals were grown from 2.4 M ammonium sulfate, 0.1 M trisodium citrate, pH 5.8. The crystals belong to space group P2₁ with cell dimensions a = 38.8 Å, b = 29.4 Å, c = 40.1 Å, β = 115.7°. Three-wavelength MAD experiment was carried out at BNL X9B beamline. Data were collected to 1.6 Å resolution. Anomalous differences from the selenium edge data set were used for initial location of the single Se atom using direct methods in SHELXS. Phase refinement was carried out with MLPHARE from CCP4 1suite and SHARP, with the resulting combined figure of merit of 0.46 to 1.6 Å resolution. Following density modification with SOLOMON, the experimental electron density map was used for automated model building with ARP/wARP, which built 100 out of 106 residues. A crystal of the wild-type domain was also grown and the structure—virtually identical to the mutant—was refined at 1.5 Å resolution. The N-DCLK contains a single mixed β-sheet containing five strands and a single major α-helix (residues 84-95), which binds in the concave groove of the β-sheet. There are three short 3₁₀ helices: residues 53-55, 120-122 and 148-151. A search with the coordinates of N-DCLK conducted using DALI, indicated that the DCX domain shows distinct structural similarities to the Ras-binding domain of RalGDS, the PB1 domain of Bem1p, UBX domain of human FAF1, and ubiquitin, all members of the ubiquitin-like superfamily.