RESONANCE ASSIGNMENTS AND SOLUTION STRUCTURE OF THE 2ND RNA-BINDING DOMAIN OF SEX-LETHAL DETERMINED BY MULTIDIMENSIONAL HETERONUCLEAR MAGNETIC-RESONANCE

The RNA-binding protein Sex-lethal (Sxl) is a critical regulator of sexual differentiation and dosage compensation in Drosophila. This regulatory activity is a consequence of the ability of Sxl to bind uridine-rich RNA tracts involved in pre-mRNA splicing. Sxl contains two RNP consensus-type RNA-binding domains (RBDs). A structural study of a portion of Sxl (amino acids 199-294) containing the second RNA-binding domain(RBD-2) using multidimensional heteronuclear NMR is presented here. Nearly complete H-1, C-13, and N-15 resonance assignments have been obtained from N-15- and C-13/N-15-uniformly labeled protein. These assignments were used to analyze 3D N-15-separated NOESY and C-13/C-13-separated 4D NOESY spectra which produced 494 total and 169 long-range NOE-derived distance restraints. Along with 41 backbone dihedral restraints; these distance restraints were employed to generate an intermediate-resolution family of calculated structures, which exhibits the beta alpha beta-beta alpha beta tertiary fold found in other RBD-containing proteins. The RMSD to the average structure for the backbone atoms of residues 11-93 is 1.55 +/- 0.30 Angstrom, while the RMSD for backbone atoms involved in secondary structure is 0.76 +/- 0.14 Angstrom. A capping box [Harper, E. T., and Rose, G. D. (1993) Biochemistry 32, 7605-7609] was identified at the N-terminus of the first helix and has been characterized by short- and medium-range NOEs. Finally, significant structural similarities and differences between Sxl RBD-2 and other RBD-containing proteins are discussed.